Antibody to secreted polypeptide

ABSTRACT

The invention provides human secreted proteins (SECP) and polynucleotides which identify and encode SECP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of SECP.

This application is a Divisional of U.S. patent application Ser. No.12/585,371, filed Sep. 14, 2009, now U.S. Pat. No. 8,569,445, which is aDivisional of U.S. patent application Ser. No. 11/378,616, filed Mar.20, 2006, now U.S. Pat. No. 7,608,704, which is a Divisional of U.S.patent application Ser. No. 10/416,314, filed May 8, 2003, nowabandoned, which is the National Phase of PCT/US01/47420, filed Nov. 8,2001, and published as WO 2002/38602 on May 16, 2002, which claimspriority to U.S. Provisional Patent Application Nos. 60/247,505, filedNov. 8, 2000, 60/247,642, filed Nov. 9, 2000, 60/249,824, filed Nov. 16,2000, 60/252,824, filed Nov. 21, 2000, 60/254,305, filed Dec. 8, 2000,and 60/256,448, filed Dec. 18, 2000. The contents of these applicationsare incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to nucleic acid and amino acid sequences ofsecreted proteins and to the use of these sequences in the diagnosis,treatment, and prevention of cell proliferative,autoimmune/inflammatory, cardiovascular, neurological, and developmentaldisorders, and in the assessment of the effects of exogenous compoundson the expression of nucleic acid and amino acid sequences of secretedproteins.

BACKGROUND OF THE INVENTION

Protein transport and secretion are essential for cellular function.Protein transport is mediated by a signal peptide located at the aminoterminus of the protein to be transported or secreted. The signalpeptide is comprised of about ten to twenty hydrophobic amino acidswhich target the nascent protein from the ribosome to a particularmembrane bound compartment such as the endoplasmic reticulum (ER).Proteins targeted to the ER may either proceed through the secretorypathway or remain in any of the secretory organelles such as the ER,Golgi apparatus, or lysosomes. Proteins that transit through thesecretory pathway are either secreted into the extracellular space orretained in the plasma membrane. Proteins that are retained in theplasma membrane contain one or more transmembrane domains, eachcomprised of about 20 hydrophobic amino acid residues. Secreted proteinsare generally synthesized as inactive precursors that are activated bypost-translational processing events during transit through thesecretory pathway. Such events include glycosylation, proteolysis, andremoval of the signal peptide by a signal peptidase. Other events thatmay occur during protein transport include chaperone-dependent unfoldingand folding of the nascent protein and interaction of the protein with areceptor or pore complex. Examples of secreted proteins with aminoterminal signal peptides are discussed below and include proteins withimportant roles in cell-to-cell signaling. Such proteins includetransmembrane receptors and cell surface markers, extracellular matrixmolecules, cytokines, hormones, growth and differentiation factors,enzymes, neuropeptides, vasomediators, cell surface markers, and antigenrecognition molecules. (Reviewed in Alberts, B. et al. (1994) MolecularBiology of The Cell, Garland Publishing, New York, N.Y., pp. 557-560,582-592.)

Cell surface markers include cell surface antigens identified onleukocytic cells of the immune system. These antigens have beenidentified using systematic, monoclonal antibody (mAb)-based “shot gun”techniques. These techniques have resulted in the production of hundredsof mAbs directed against unknown cell surface leukocytic antigens. Theseantigens have been grouped into “clusters of differentiation” based oncommon immunocytochemical localization patterns in variousdifferentiated and undifferentiated leukocytic cell types. Antigens in agiven cluster are presumed to identify a single cell surface protein andare assigned a “cluster of differentiation” or “CD” designation. Some ofthe genes encoding proteins identified by CD antigens have been clonedand verified by standard molecular biology techniques. CD antigens havebeen characterized as both transmembrane proteins and cell surfaceproteins anchored to the plasma membrane via covalent attachment tofatty acid-containing glycolipids such as glycosylphosphatidylinositol(GPI). (Reviewed in Barclay, A. N. et al. (1995) The Leucocyte AntigenFacts Book, Academic Press, San Diego, Calif., pp. 17-20.)

Matrix proteins (MPs) are transmembrane and extracellular proteins whichfunction in formation, growth, remodeling, and maintenance of tissuesand as important mediators and regulators of the inflammatory response.The expression and balance of MPs may be perturbed by biochemicalchanges that result from congenital, epigenetic, or infectious diseases.In addition, MPs affect leukocyte migration, proliferation,differentiation, and activation in the immune response. MPs arefrequently characterized by the presence of one or more domains whichmay include collagen-like domains, EGF-like domains, immunoglobulin-likedomains, and fibronectin-like domains. In addition, MPs may be heavilyglycosylated and may contain an Arginine-Glycine-Aspartate (RGD)tripeptide motif which may play a role in adhesive interactions. MPsinclude extracellular proteins such as fibronectin, collagen, galectin,vitronectin and its proteolytic derivative somatomedin B; and celladhesion receptors such as cell adhesion molecules (CAMs), cadherins,and integrins. (Reviewed in Ayad, S. et al. (1994) The ExtracellularMatrix Facts Book, Academic Press, San Diego, Calif., pp. 2-16;Ruoslahti, E. (1997) Kidney Int. 51:1413-1417; Sjaastad, M. D. andNelson, W. J. (1997) BioEssays 19:47-55.)

Peroxidasin is a Drosophila protein that contains both peroxidase andextracellular matrix motifs. The 1512 amino acid peroxidasin proteincontains a peroxidase domain homologous to human myeloperoxidase andeosiniphil peroxidase, as well as six leucine-rich repeats, fourimmunoglobulin domains, and a region of thrombospondin/procollagenhomology. Peroxidasin is secreted by hemocytes as they spread throughoutthe developing Drosophila embryo. The protein is thought to function inextracellular matrix consolidation, phagocytosis, and defense (Nelson,R. E. (1994) EMBO J. 13:3438-3447). A human homolog of the Drosophilaperoxidasin gene was recently found to be upregulated in a colon cancercell line undergoing p53 tumor suppressor-dependent apoptosis, and thusmay play a role in the mechanisms of p53-dependent apoptosis (Horikoshi,N. et al. (1999) Biochem. Biophy. Res. Common. 261:864-869).

Mucins are highly glycosylated glycoproteins that are the majorstructural component of the mucus gel. The physiological functions ofmucins are cytoprotection, mechanical protection, maintenance ofviscosity in secretions, and cellular recognition. MUC6 is a humangastric mucin that is also found in gall bladder, pancreas, seminalvesicles, and female reproductive tract (Toribara, N. W. et al. (1997)J. Biol. Chem. 272:16398-16403). The MUC6 gene has been mapped to humanchromosome 11 (Toribara, N. W. et al. (1993) J. Biol. Chem.268:5879-5885). Hemomucin is a novel Drosophila surface mucin that maybe involved in the induction of antibacterial effector molecules(Theopold, U. et al. (1996) J. Biol. Chem. 217:12708-12715).

Tuftelins are one of four different enamel matrix proteins that havebeen identified so far. The other three known enamel matrix proteins arethe amelogenins, enamelin and ameloblastin. Assembly of the enamelextracellular matrix from these component proteins is believed to becritical in producing a matrix competent to undergo mineral replacement.(Paine, C. T. et al. (1998) Connect Tissue Res. 38:257-267). TuftelinmRNA has been found to be expressed in human ameloblastoma tumor, anon-mineralized odontogenic tumor (Deutsch, D. et al. (1998) Connect.Tissue Res. 39:177-184).

Olfactomedin-related proteins are extracellular matrix, secretedglycoproteins with conserved C-terminal motifs. They are expressed in awide variety of tissues and in broad range of species, fromCaenorhabditis elegans to Homo sapiens. Olfactomedin-related proteinscomprise a gene family with at least 5 family members in humans. One ofthe five, TIGR/myocilin protein, is expressed in the eye and isassociated with the pathogenesis of glaucoma (Kulkarni, N. H. et al.(2000) Genet. Res. 76:41-50). Research by Yokoyama et al. (1996) found a135-amino acid protein, termed AMY, having 96% sequence identity withrat neuronal olfactomedin-releated ER localized protein in aneuroblastoma cell line cDNA library, suggesting an essential role forAMY in nerve tissue (Yokoyama, M. et al. (1996) DNA Res. 3:311-320).Neuron-specific olfactomedin-related glycoproteins isolated from ratbrain cDNA libraries show strong sequence similarity with olfactomedin.This similarity is suggestive of a matrix-related function of theseglycoproteins in neurons and neurosecretory cells (Danielson, P. E. etal. (1994) J. Neurosci. Res. 38:468-478).

Mac-2 binding protein is a 90-10 serum protein (90K), a secretedglycoprotein isolated from both the human breast carcinoma cell lineSK-BR-3, and human breast milk. It specifically binds to a humanmacrophage-associated lectin, Mac-2. Structurally, the mature protein is567 amino acids in length and is proceeded by an 18-amino acid leader.There are 16 cysteines and seven potential N-linked glycosylation sites.The first 106 amino acids represent a domain very similar to an ancientprotein superfamily defined by a macrophage scavenger receptorcysteine-rich domain (Koths, K. et al. (1993) J. Biol. Chem.268:14245-14249). 90K is elevated in the serum of subpopulations of AIDSpatients and is expressed at varying levels in primary tumor samples andtumor cell lines. Ullrich et al. (1994) have demonstrated that 90Kstimulates host defense systems and can induce interleukin-2 secretion.This immune stimulation is proposed to be a result of oncogenictransformation, viral infection or pathogenic invasion (Ullrich, A., etal. (1994) J. Biol. Chem. 269:18401-18407).

Semaphorins are a large group of axonal guidance molecules consisting ofat least 30 different members and are found in vertebrates,invertebrates, and even certain viruses. All semaphorins contain thesema domain which is approximately 500 amino acids in length.Neuropilin, a semaphorin receptor, has been shown to promote neuriteoutgrowth in vitro. The extracellular region of neuropilins consists ofthree different domains: CUB, discoidin, and MAM domains. The CUB andthe MAM motifs of neuropilin have been suggested to have roles inprotein-protein interactions and are thought to be involved in thebinding of semaphorins through the sema and the C-terminal domains(reviewed in Raper, J. A. (2000) Curr. Opin. Neurobiol. 10:88-94).Plexins are neuronal cell surface molecules that mediate cell adhesionvia a homophilic binding mechanism in the presence of calcium ions.Plexins have been shown to be expressed in the receptors and neurons ofparticular sensory systems (Ohta, K. et al. (1995) Cell 14:1189-1199).There is evidence that suggests that some plexins function to controlmotor and CNS axon guidance in the developing nervous system. Plexins,which themselves contain complete semaphorin domains, may be both theancestors of classical semaphorins and binding partners for semaphorins(Winberg, M. L. et al (1998) Cell 95:903-916).

Human pregnancy-specific beta 1-glycoprotein (PSG) is a family ofclosely related glycoproteins of molecular weights of 72 KDa, 64 KDa, 62KDa, and 54 KDa. Together with the carcinoembryonic antigen, theycomprise a subfamily within the immunoglobulin superfamily (Plouzek, C.A. and Chou, J. Y. (1991) Endocrinology 129:950-958) Differentsubpopulations of PSG have been found to be produced by the trophoblastsof the human placenta, and the amnionic and chorionic membranes(Plouzek, C. A. et al. (1993) Placenta 14:277-285).

Autocrine motility factor (AMF) is one of the motility cytokinesregulating tumor cell migration; therefore identification of thesignaling pathway coupled with it has critical importance. Autocrinemotility factor receptor (AMFR) expression has been found to beassociated with tumor progression in thymoma (Ohta Y. et al. (2000) Int.J. Oncol. 17:259-264). AMER is a cell surface glycoprotein of molecularweight 78 KDa.

Hormones are secreted molecules that travel through the circulation andbind to specific receptors on the surface of, or within, target cells.Although they have diverse biochemical compositions and mechanisms ofaction, hormones can be grouped into two categories. One categoryincludes small lipophilic hormones that diffuse through the plasmamembrane of target cells, bind to cytosolic or nuclear receptors, andform a complex that alters gene expression. Examples of these moleculesinclude retinoic acid, thyroxine, and the cholesterol-derived steroidhormones such as progesterone, estrogen, testosterone, cortisol, andaldosterone. The second category includes hydrophilic hormones thatfunction by binding to cell surface receptors that transduce signalsacross the plasma membrane. Examples of such hormones include amino acidderivatives such as catecholamines (epinephrine, norepinephrine) andhistamine, and peptide hormones such as glucagon, insulin, gastrin,secretin, cholecystokinin, adrenocorticotropic hormone, folliclestimulating hormone, luteinizing hormone, thyroid stimulating hormone,and vasopressin. (See, for example, Lodish et al. (1995) Molecular CellBiology, Scientific American Books Inc., New York, N.Y., pp. 856-864.)

Pro-opiomelanocortin (POMC) is the precursor polypeptide ofcorticotropin (ACTH), a hormone synthesized by the anterior pituitarygland, which functions in the stimulation of the adrenal cortex. POMC isalso the precursor polypeptide of the hormone beta-lipotropin(beta-LPH). Each hormone includes smaller peptides with distinctbiological activities: alpha-melanotropin (alpha-MSH) andcorticotropin-like intermediate lobe peptide (CLIP) are formed fromACTH; gamma-lipotropin (gamma-LPH) and beta-endorphin are peptidecomponents of beta-LPH; while beta-MSH is contained within gamma-LPH.Adrenal insufficiency due to ACTH deficiency, resulting from a geneticmutation in exons 2 and 3 of POMC results in an endocrine disordercharacterized by early-onset obesity, adrenal insufficiency, and redhair pigmentation (Chretien, M. et al. (1979) Canad. J. Biochem.57:1111-1121; Krude, H. et al. (1998) Nature Genet. 19:155-157; OnlineMendelian Inheritance in Man (OMIM) 176830).

Growth and differentiation factors are secreted proteins which functionin intercellular communication. Some factors require oligomerization orassociation with membrane proteins for activity. Complex interactionsamong these factors and their receptors trigger intracellular signaltransduction pathways that stimulate or inhibit cell division, celldifferentiation, cell signaling, and cell motility. Most growth anddifferentiation factors act on cells in their local environment(paracrine signaling). There are three broad classes of growth anddifferentiation factors. The first class includes the large polypeptidegrowth factors such as epidermal growth factor, fibroblast growthfactor, transforming growth factor, insulin-like growth factor, andplatelet-derived growth factor. The second class includes thehematopoietic growth factors such as the colony stimulating factors(CSFs). Hematopoietic growth factors stimulate the proliferation anddifferentiation of blood cells such as B-lymphocytes, T-lymphocytes,erythrocytes, platelets, eosinophils, basophils, neutrophils,macrophages, and their stem cell precursors. The third class includessmall peptide factors such as bombesin, vasopressin, oxytocin,endothelin, transferrin, angiotensin II, vasoactive intestinal peptide,and bradykinin, which function as hormones to regulate cellularfunctions other than proliferation.

Growth and differentiation factors play critical roles in neoplastictransformation of cells in vitro and in tumor progression in vivo.Inappropriate expression of growth factors by tumor cells may contributeto vascularization and metastasis of tumors. During hematopoiesis,growth factor misregulation can result in anemias, leukemias, andlymphomas. Certain growth factors such as interferon are cytotoxic totumor cells both in vivo and in vitro. Moreover, some growth factors andgrowth factor receptors are related both structurally and functionallyto oncoproteins. In addition, growth factors affect transcriptionalregulation of both proto-oncogenes and oncosuppressor genes. (Reviewedin Pimentel, E. (1994) Handbook of Growth Factors, CRC Press, Ann Arbor,Mich., pp. 1-9.)

The Slit protein, first identified in Drosophila, is critical in centralnervous system midline formation and potentially in nervous tissuehistogenesis and axonal pathfinding. Itoh et al. ((1998) Brain Res. Mol.Brain. Res. 62:175-186) have identified mammalian homologues of the slitgene (human Slit-1, Slit-2, Slit-3 and rat Slit-1). The encoded proteinsare putative secreted proteins containing EGF-like motifs andleucine-rich repeats, both of which are conserved protein-proteininteraction domains. Slit-1, -2, and -3 mRNAs are expressed in thebrain, spinal cord, and thyroid, respectively (Itoh, A. et al., supra).The Slit family of proteins are indicated to be functional ligands ofglypican-1 in nervous tissue and it is suggested that their interactionsmay be critical in certain stages during central nervous systemhistogenesis (Liang, Y. et al., (1999) J. Biol. Chem. 274:17885-17892).

Neuropeptides and vasomediators (NP/VM) comprise a large family ofendogenous signaling molecules. Included in this family areneuropeptides and neuropeptide hormones such as bombesin, neuropeptideY, neurotensin, neuromedin N, melanocortins, opioids, galanin,somatostatin, tachykinins, urotensin II and related peptides involved insmooth muscle stimulation, vasopressin, vasoactive intestinal peptide,and circulatory system-borne signaling molecules such as angiotensin,complement, calcitonin, endothelins, formyl-methionyl peptides,glucagon, cholecystokinin and gastrin. NP/VMs can transduce signalsdirectly, modulate the activity or release of other neurotransmittersand hormones, and act as catalytic enzymes in cascades. The effects ofNP/VMs range from extremely brief to long-lasting. (Reviewed in Martin,C. R. et al. (1985) Endocrine Physiology, Oxford University Press, NewYork, N.Y., pp. 57-62.)

NP/VMs are involved in numerous neurological and cardiovasculardisorders. For example, neuropeptide Y is involved in hypertension,congestive heart failure, affective disorders, and appetite regulation.Somatostatin inhibits secretion of growth hormone and prolactin in theanterior pituitary, as well as inhibiting secretion in intestine,pancreatic acinar cells, and pancreatic beta-cells. A reduction insomatostatin levels has been reported in Alzheimer's disease andParkinson's disease. Vasopressin acts in the kidney to increase waterand sodium absorption, and in higher concentrations stimulatescontraction of vascular smooth muscle, platelet activation, and glycogenbreakdown in the liver. Vasopressin and its analogues are usedclinically to treat diabetes insipidus. Endothelin and angiotensin areinvolved in hypertension, and drugs, such as captopril, which reduceplasma levels of angiotensin, are used to reduce blood pressure (Watson,S, and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book,Academic Press, San Diego Calif., pp. 194; 252; 284; 55; 111).

Neuropeptides have also been shown to have roles in nociception (pain).Vasoactive intestinal peptide appears to play an important role inchronic neuropathic pain. Nociceptin, an endogenous ligand for theopioid receptor-like 1 receptor, is thought to have a predominantlyanti-nociceptive effect, and has been shown to have analgesic propertiesin different animal models of tonic or chronic pain (Dickinson, T. andFleetwood-Walker, S. M. (1998) Trends Pharmacol. Sci. 19:346-348).

Other proteins that contain signal peptides include secreted proteinswith enzymatic activity. Such activity includes, for example,oxidoreductase/dehydrogenase activity, transferase activity, hydrolaseactivity, lyase activity, isomerase activity, or ligase activity. Forexample, matrix metalloproteinases are secreted hydrolytic enzymes thatdegrade the extracellular matrix and thus play an important role intumor metastasis, tissue morphogenesis, and arthritis (Reponen, P. etal. (1995) Dev. Dyn. 202:388-396; Firestein, G. S. (1992) Curr. Opin.Rheumatol. 4:348-354; Ray, J. M. and Stetler-Stevenson, W. G. (1994)Eur. Respir. J. 7:2062-2072; and Mignatti, P. and Rifkin, D. B. (1993)Physiol. Rev. 73:161-195). Additional examples are the acetyl-CoAsynthetases which activate acetate for use in lipid synthesis or energygeneration (Luong, A. et al. (2000) J. Biol. Chem. 275:26458-26466). Theresult of acetyl-CoA synthetase activity is the formation of acetyl-CoAfrom acetate and CoA. Acetyl-CoA sythetases share a region of sequencesimilarity identified as the AMP-binding domain signature. Acetyl-CoAsynthetase has been shown to be associated with hypertension (H. Toh(1991) Protein Seq. Data Anal. 4:111-117; and Iwai, N. et al., (1994)Hypertension 23:375-380).

A number of isomerases catalyze steps in protein folding,phototransduction, and various anabolic and catabolic pathways. Oneclass of isomerases is known as peptidyl-prolyl cis-trans isomerases(PPIases). PPIases catalyze the cis to trans isomerization of certainproline imidic bonds in proteins. Two families of PPIases are the FK506binding proteins (FKBPs), and cyclophilins (CyPs). FKBPs bind the potentimmunosuppressants FK506 and rapamycin, thereby inhibiting signalingpathways in T-cells. Specifically, the PPIase activity of FKBPs isinhibited by binding of FK506 or rapamycin. There are five members ofthe FKBP family which are named according to their calculated molecularmasses (FKBP12, FKBP13, FKBP25, FKBP52, and FKBP65), and localized todifferent regions of the cell where they associate with differentprotein complexes (Coss, M. et al. (1995) J. Biol. Chem.270:29336-29341; Schreiber, S. L. (1991) Science 251:283-287).

The peptidyl-prolyl isomerase activity of CyP may be part of thesignaling pathway that leads to T-cell activation. CyP isomeraseactivity is associated with protein folding and protein trafficking, andmay also be involved in assembly/disassembly of protein complexes andregulation of protein activity. For example, in Drosophila, the CyPNinaA is required for correct localization of rhodopsins, while amammalian CyP (Cyp40) is part of the Hsp90/Hsc70 complex that bindssteroid receptors. The mammalian CypA has been shown to bind the gagprotein from human immunodeficiency virus 1 (HIV-1), an interaction thatcan be inhibited by cyclosporin. Since cyclosporin has potent anti-HIV-1activity, CypA may play an essential function in HIV-1 replication.Finally, Cyp40 has been shown to bind and inactivate the transcriptionfactor c-Myb, an effect that is reversed by cyclosporin. This effectimplicates CyPs in the regulation of transcription, transformation, anddifferentiation (Bergsma, D. J. et al (1991) J. Biol. Chem.266:23204-23214; Hunter, T. (1998) Cell 92: 141-143; and Leverson, J. D.and Ness, S. A. (1998) Mol. Cell. 1:203-211).

Gamma-carboxyglutamic acid (Gla) proteins rich in proline (PRGPs) aremembers of a family of vitamin K-dependent single-pass integral membraneproteins. These proteins are characterized by an extracellular aminoterminal domain of approximately 45 amino acids rich in Gla. Theintracellular carboxyl terminal region contains one or two copies of thesequence PPXY, a motif present in a variety of proteins involved in suchdiverse cellular functions as signal transduction, cell cycleprogression, and protein turnover (Kulman, J. D. et al., (2001) Proc.Natl. Acad. Sci. U.S.A. 98:1370-1375). The process of post-translationalmodification of glutamic residues to form Gla is Vitamin K-dependentcarboxylation. Proteins which contain Gla include plasma proteinsinvolved in blood coagulation. These proteins are prothrombin, proteinsC, S, and Z, and coagulation factors VII, IX, and X. Osteocalcin(bone-Gla protein, BGP) and matrix Gla-protein (MGP) also contain Gla(Friedman, P. A., and C. T. Przysiecki (1987) Int. J. Biochem. 19:1-7;C. Vermeer (1990) Biochem. J. 266:625-636).

The Drosophila sp. gene crossveinless 2 is characterized as having aputative signal or transmembrane sequence, and a partial Von WillebrandFactor D domain similar to those domains known to regulate the formationof intramolecular and intermolecular bonds and five cysteine-richdomains, known to bind BMP-like (bone morphogenetic proteins) ligands.These features suggest that crossveinless 2 may act extracellularly orin the secretory pathway to directly potentiate ligand signaling andhence, involvement in the BMP-like signaling pathway known to play arole in vein specification (Conley, C. A. et al., (2000) Development127:3947-3959). The dorsal-ventral patterning in both vertebrate andDrosophila embryos requires a conserved system of extracellular proteinsto generate a positional informational gradient.

The discovery of new secreted proteins, and the polynucleotides encodingthem, satisfies a need in the art by providing new compositions whichare useful in the diagnosis, prevention, and treatment of cellproliferative, autoimmune/inflammatory, cardiovascular, neurological,and developmental disorders, and in the assessment of the effects ofexogenous compounds on the expression of nucleic acid and amino acidsequences of secreted proteins.

SUMMARY OF THE INVENTION

The invention features purified polypeptides, secreted proteins,referred to collectively as “SECP” and individually as “SECP-1,”“SECP-2,” “SECP-3,” “SECP-4,” “SECP-5,” “SECP-6,” “SECP-7,” “SECP-8,”“SECP-9,” “SECP-10,” “SECP-11,” “SECP-12,” “SECP-13,” “SECP-14“SECP-15,” “SECP-16,” “SECP-17,” “SECP-18,” “SECP-19,” “SECP-20,”“SECP-21,” “SECP-22,” “SECP-23,” “SECP-24,” “SECP-25,” “SECP-26,”“SECP-27,” “SECP-28,”, “SECP-29,” “SECP-30,” “SECP-31,” “SECP-32,”“SECP-33,” “SECP-34,” “SECP-35,” “SECP-36,” “SECP-37,” “SECP-38,”“SECP-39,” “SECP-40,” “SECP-41,” “SECP-42,” “SECP-43 “SECP-44,”“SECP-45,” “SECP-46,” “SECP-47,” “SECP-48,” “SECP-49,” “SECP-50,”“SECP-51,” “SECP-52,” “SECP-53,” “SECP-54,” “SECP-55,” “SECP-56,”“SECP-57,” “SECP-58,” “SECP-59,” “SECP-60,” “SECP-61,” “SECP-62,” and“SECP-63.” In one aspect, the invention provides an isolated polypeptideselected from the group consisting of a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ IDNO:1-63, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-63, c) a biologically activefragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-63, and d) an immunogenic fragmentof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-63. In one alternative, the invention providesan isolated polypeptide comprising the amino acid sequence of SEQ IDNO:1-63.

The invention further provides an isolated polynucleotide encoding apolypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-63, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-63, c) a biologically activefragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-63, and d) an immunogenic fragmentof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-63. In one alternative, the polynucleotideencodes a polypeptide selected from the group consisting of SEQ IDNO:1-63. In another alternative, the polynucleotide is selected from thegroup consisting of SEQ ID NO:64-126.

Additionally, the invention provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-63, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:1-63, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-63, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-63. In onealternative, the invention provides a cell transformed with therecombinant polynucleotide. In another alternative, the inventionprovides a transgenic organism comprising the recombinantpolynucleotide.

The invention also provides a method for producing a polypeptideselected from the group consisting of a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ IDNO:1-63, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-63, c) a biologically activefragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-63, and d) an immunogenic fragmentof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-63. The method comprises a) culturing a cellunder conditions suitable for expression of the polypeptide, whereinsaid cell is transformed with a recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide encoding thepolypeptide, and b) recovering the polypeptide so expressed.

Additionally, the invention provides an isolated antibody whichspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-63, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical to anamino acid sequence selected from the group consisting of SEQ IDNO:1-63, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-63, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-63.

The invention further provides an isolated polynucleotide selected fromthe group consisting of a) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:64-126, b) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:64-126, c) a polynucleotide complementaryto the polynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). In onealternative, the polynucleotide comprises at least 60 contiguousnucleotides.

Additionally, the invention provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide selected from the group consisting of a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:64-126, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:64-126, c) a polynucleotide complementary to the polynucleotide ofa), d) a polynucleotide complementary to the polynucleotide of b), ande) an RNA equivalent of a)-d). The method comprises a) hybridizing thesample with a probe comprising at least 20 contiguous nucleotidescomprising a sequence complementary to said target polynucleotide in thesample, and which probe specifically hybridizes to said targetpolynucleotide, under conditions whereby a hybridization complex isformed between said probe and said target polynucleotide or fragmentsthereof, and b) detecting the presence or absence of said hybridizationcomplex, and optionally, if present, the amount thereof. In onealternative, the probe comprises at least 60 contiguous nucleotides.

The invention further provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide selected from the group consisting of a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:64-126, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:64-126, c) a polynucleotide complementary to the polynucleotide ofa), d) a polynucleotide complementary to the polynucleotide of b), ande) an RNA equivalent of a)-d). The method comprises a) amplifying saidtarget polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.

The invention further provides a composition comprising an effectiveamount of a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-63, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:1-63, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-63, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-63, and apharmaceutically acceptable excipient. In one embodiment, thecomposition comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-63. The invention additionally provides amethod of treating a disease or condition associated with decreasedexpression of functional SECP, comprising administering to a patient inneed of such treatment the composition.

The invention also provides a method for screening a compound foreffectiveness as an agonist of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-63, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-63, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-63, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-63. The method comprises a) exposing a sample comprising thepolypeptide to a compound, and b) detecting agonist activity in thesample. In one alternative, the invention provides a compositioncomprising an agonist compound identified by the method and apharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with decreased expression of functional SECP, comprisingadministering to a patient in need of such treatment the composition.

Additionally, the invention provides a method for screening a compoundfor effectiveness as an antagonist of a polypeptide selected from thegroup consisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-63, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-63, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-63, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-63. The method comprises a) exposing a sample comprising thepolypeptide to a compound, and b) detecting antagonist activity in thesample. In one alternative, the invention provides a compositioncomprising an antagonist compound identified by the method and apharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with overexpression of functional SECP, comprisingadministering to a patient in need of such treatment the composition.

The invention further provides a method of screening for a compound thatspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-63, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical to anamino acid sequence selected from the group consisting of SEQ IDNO:1-63, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-63, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-63. Themethod comprises a) combining the polypeptide with at least one testcompound under suitable conditions, and b) detecting binding of thepolypeptide to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide.

The invention further provides a method of screening for a compound thatmodulates the activity of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-63, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-63, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-63, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-63. The method comprises a) combining the polypeptide with at leastone test compound under conditions permissive for the activity of thepolypeptide, b) assessing the activity of the polypeptide in thepresence of the test compound, and c) comparing the activity of thepolypeptide in the presence of the test compound with the activity ofthe polypeptide in the absence of the test compound, wherein a change inthe activity of the polypeptide in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptide.

The invention further provides a method for screening a compound foreffectiveness in altering expression of a target polynucleotide, whereinsaid target polynucleotide comprises a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:64-126, the method comprising a)exposing a sample comprising the target polynucleotide to a compound,and b) detecting altered expression of the target polynucleotide.

The invention further provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide selected from thegroup consisting of i) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:64-126, ii) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:64-126, iii) a polynucleotide having asequence complementary to i), iv) a polynucleotide complementary to thepolynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridizationoccurs under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide selected from the group consisting ofi) a polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:64-126, ii) a polynucleotidecomprising a naturally occurring polynucleotide sequence at least 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:64-126, iii) a polynucleotide complementary tothe polynucleotide of i), iv) a polynucleotide complementary to thepolynucleotide of ii), and v) an RNA equivalent of i)-iv).Alternatively, the target polynucleotide comprises a fragment of apolynucleotide sequence selected from the group consisting of i)-v)above; c) quantifying the amount of hybridization complex; and d)comparing the amount of hybridization complex in the treated biologicalsample with the amount of hybridization complex in an untreatedbiological sample, wherein a difference in the amount of hybridizationcomplex in the treated biological sample is indicative of toxicity ofthe test compound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide sequences of the present invention.

Table 2 shows the GenBank identification number and annotation of thenearest GenBank homolog, for polypeptides of the invention. Theprobability scores for the matches between each polypeptide and itshomolog(s) are also shown.

Table 3 shows structural features of polypeptide sequences of theinvention, including predicted motifs and domains, along with themethods, algorithms, and searchable databases used for analysis of thepolypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used toassemble polynucleotide sequences of the invention, along with selectedfragments of the polynucleotide sequences.

Table 5 shows the representative cDNA library for polynucleotides of theinvention.

Table 6 provides an appendix which describes the tissues and vectorsused for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyze thepolynucleotides and polypeptides of the invention, along with applicabledescriptions, references, and threshold parameters.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular machines, materials and methods described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

DEFINITIONS

“SECP” refers to the amino acid sequences of substantially purified SECPobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine, and human, and from any source,whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics thebiological activity of SECP. Agonists may include proteins, nucleicacids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of SECP either by directlyinteracting with SECP or by acting on components of the biologicalpathway in which SECP participates.

An “allelic variant” is an alternative form of the gene encoding SECP.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. A gene may have none,one, or many allelic variants of its naturally occurring form. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding SECP include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as SECP or a polypeptide with atleast one functional characteristic of SECP. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingSECP, and improper or unexpected hybridization to allelic variants, witha locus other than the normal chromosomal locus for the polynucleotidesequence encoding SECP. The encoded protein may also be “altered,” andmay contain deletions, insertions, or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent SECP. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of SECP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, and positively charged amino acids may include lysine andarginine. Amino acids with uncharged polar side chains having similarhydrophilicity values may include: asparagine and glutamine; and serineand threonine. Amino acids with uncharged side chains having similarhydrophilicity values may include: leucine, isoleucine, and valine;glycine and alanine; and phenylalanine and tyrosine.

The terms “amino acid” and “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules.Where “amino acid sequence” is recited to refer to a sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

“Amplification” relates to the production of additional copies of anucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

The term “antagonist” refers to a molecule which inhibits or attenuatesthe biological activity of SECP. Antagonists may include proteins suchas antibodies, nucleic acids, carbohydrates, small molecules, or anyother compound or composition which modulates the activity of SECPeither by directly interacting with SECP or by acting on components ofthe biological pathway in which SECP participates.

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant. Antibodies that bind SECPpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that region of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (particular regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “aptamer” refers to a nucleic acid or oligonucleotide moleculethat binds to a specific molecular target. Aptamers are derived from anin vitro evolutionary process (e.g., SELEX (Systematic Evolution ofLigands by EXponential Enrichment), described in U.S. Pat. No.5,270,163), which selects for target-specific aptamer sequences fromlarge combinatorial libraries. Aptamer compositions may bedouble-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′-OH group of a ribonucleotidemay be replaced by 2′-F or 2′—NH₂), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in blood.Aptamers may be conjugated to other molecules, e.g., a high molecularweight carrier to slow clearance of the aptamer from the circulatorysystem. Aptamers may be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody,E. N. and L. Gold (2000) J. Biotechnol. 74:5-13.)

The term “intramer” refers to an aptamer which is expressed in vivo. Forexample, a vaccinia virus-based RNA expression system has been used toexpress specific RNA aptamers at high levels in the cytoplasm ofleukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA96:3606-3610).

The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA,or other left-handed nucleotide derivatives or nucleotide-likemolecules. Aptamers containing left-handed nucleotides are resistant todegradation by naturally occurring enzymes, which normally act onsubstrates containing right-handed nucleotides.

The term “antisense” refers to any composition capable of base-pairingwith the “sense” (coding) strand of a specific nucleic acid sequence.Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA);oligonucleotides having modified backbone linkages such asphosphorothioates, methylphosphonates, or benzylphosphonates;oligonucleotides having modified sugar groups such as 2′-methoxyethylsugars or 2′-methoxyethoxy sugars; or oligonucleotides having modifiedbases such as 5-methyl cytosine, 2′-deoxyuracil, or7-deaza-2′-deoxyguanosine. Antisense molecules may be produced by anymethod including chemical synthesis or transcription. Once introducedinto a cell, the complementary antisense molecule base-pairs with anaturally occurring nucleic acid sequence produced by the cell to formduplexes which block either transcription or translation. Thedesignation “negative” or “minus” can refer to the antisense strand, andthe designation “positive” or “plus” can refer to the sense strand of areference DNA molecule.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic SECP, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing. For example,5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

A “composition comprising a given polynucleotide sequence” and a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encoding SECPor fragments of SECP may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beensubjected to repeated DNA sequence analysis to resolve uncalled bases,extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.)in the 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from one or more overlapping cDNA, EST, or genomic DNAfragments using a computer program for fragment assembly, such as theGELVIEW fragment assembly system (GCG, Madison Wis.) or Phrap(University of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that arepredicted to least interfere with the properties of the originalprotein, i.e., the structure and especially the function of the proteinis conserved and not significantly changed by such substitutions. Thetable below shows amino acids which may be substituted for an originalamino acid in a protein and which are regarded as conservative aminoacid substitutions.

Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln,His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser,Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide can include, forexample, replacement of hydrogen by an alkyl, acyl, hydroxyl, or aminogroup. A derivative polynucleotide encodes a polypeptide which retainsat least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that iscapable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

“Differential expression” refers to increased or upregulated; ordecreased, downregulated, or absent gene or protein expression,determined by comparing at least two different samples. Such comparisonsmay be carried out between, for example, a treated and an untreatedsample, or a diseased and a normal sample.

“Exon shuffling” refers to the recombination of different coding regions(exons). Since an exon may represent a structural or functional domainof the encoded protein, new proteins may be assembled through the novelreassortment of stable substructures, thus allowing acceleration of theevolution of new protein functions.

A “fragment” is a unique portion of SECP or the polynucleotide encodingSECP which is identical in sequence to but shorter in length than theparent sequence. A fragment may comprise up, to the entire length of thedefined sequence, minus one nucleotide/amino acid residue. For example,a fragment may comprise from 5 to 1000 contiguous nucleotides or aminoacid residues. A fragment used as a probe, primer, antigen, therapeuticmolecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25,30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotidesor amino acid residues in length. Fragments may be preferentiallyselected from certain regions of a molecule. For example, a polypeptidefragment may comprise a certain length of contiguous amino acidsselected from the first 250 or 500 amino acids (or first 25% or 50%) ofa polypeptide as shown in a certain defined sequence. Clearly theselengths are exemplary, and any length that is supported by thespecification, including the Sequence Listing, tables, and figures, maybe encompassed by the present embodiments.

A fragment of SEQ ID NO:64-126 comprises a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:64-126,for example, as distinct from any other sequence in the genome fromwhich the fragment was obtained. A fragment of SEQ ID NO:64-126 isuseful, for example, in hybridization and amplification technologies andin analogous methods that distinguish SEQ ID NO:64-126 from relatedpolynucleotide sequences. The precise length of a fragment of SEQ IDNO:64-126 and the region of SEQ ID NO:64-126 to which the fragmentcorresponds are routinely determinable by one of ordinary skill in theart based on the intended purpose for the fragment.

A fragment of SEQ ID NO:1-63 is encoded by a fragment of SEQ IDNO:64-126. A fragment of SEQ ID NO:1-63 comprises a region of uniqueamino acid sequence that specifically identifies SEQ ID NO:1-63. Forexample, a fragment of SEQ ID NO:1-63 is useful as an immunogenicpeptide for the development of antibodies that specifically recognizeSEQ ID NO:1-63. The precise length of a fragment of SEQ ID NO:1-63 andthe region of SEQ ID NO:1-63 to which the fragment corresponds areroutinely determinable by one of ordinary skill in the art based on theintended purpose for the fragment.

A “full length” polynucleotide sequence is one containing at least atranslation initiation codon (e.g., methionine) followed by an openreading frame and a translation termination codon. A “full length”polynucleotide sequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, interchangeably, sequenceidentity, between two or more polynucleotide sequences or two or morepolypeptide sequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing the default parameters of the CLUSTAL V algorithm as incorporatedinto the MEGALIGN version 3.12e sequence alignment program. This programis part of the LASERGENE software package, a suite of molecularbiological analysis programs (DNASTAR, Madison Wis.). CLUSTAL V isdescribed in Higgins, D. G. and P. M. Sharp (1989) CABIOS 5:151-153 andin Higgins, D. G. et al. (1992) CABIOS 8:189-191. For pairwisealignments of polynucleotide sequences, the default parameters are setas follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4.The “weighted” residue weight table is selected as the default. Percentidentity is reported by CLUSTAL V as the “percent similarity” betweenaligned polynucleotide sequences.

Alternatively, a suite of commonly used and freely available sequencecomparison algorithms is provided by the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), whichis available from several sources, including the NCBI, Bethesda, Md.,and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLASTsoftware suite includes various sequence analysis programs including“blastn,” that is used to align a known polynucleotide sequence withother polynucleotide sequences from a variety of databases. Alsoavailable is a tool called “BLAST 2 Sequences” that is used for directpairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” canbe accessed and used interactively athttp://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” toolcan be used for both blastn and blastp (discussed below). BLAST programsare commonly used with gap and other parameters set to default settings.For example, to compare two nucleotide sequences, one may use blastnwith the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set atdefault parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: −2

Open Gap: 5 and Extension Gap: 2 penalties

Gap x drop-off: 50

Expect: 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures, or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table. As with polynucleotidealignments, the percent identity is reported by CLUSTAL V as the“percent similarity” between aligned polypeptide sequence pairs.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp setat default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Open Gap: 11 and Extension Gap: 1 penalties

Gap x drop-off: 50

Expect: 10

Word Size: 3

Filter: on

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

The term “humanized antibody” refers to an antibody molecule in whichthe amino acid sequence in the non-antigen binding regions has beenaltered so that the antibody more closely resembles a human antibody,and still retains its original binding ability.

“Hybridization” refers to the process by which a polynucleotide strandanneals with a complementary strand through base pairing under definedhybridization conditions. Specific hybridization is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions and remain hybridized after the “washing” step(s). Thewashing step(s) is particularly important in determining the stringencyof the hybridization process, with more stringent conditions allowingless non-specific binding, i.e., binding between pairs of nucleic acidstrands that are not perfectly matched. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may be consistent among hybridizationexperiments, whereas wash conditions may be varied among experiments toachieve the desired stringency, and therefore hybridization specificity.Permissive annealing conditions occur, for example, at 68° C. in thepresence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/mlsheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, withreference to the temperature under which the wash step is carried out.Such wash temperatures are typically selected to be about 5° C. to 20°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. et al. (1989) Molecular Cloning:A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; specifically see volume 2, chapter 9.

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSCconcentration may be varied from about 0.1 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml.Organic solvent, such as formamide at a concentration of about 35-50%v/v, may also be used under particular circumstances, such as forRNA:DNA hybridizations. Useful variations on these wash conditions willbe readily apparent to those of ordinary skill in the art.Hybridization, particularly under high stringency conditions, may besuggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary bases. A hybridization complex may be formed insolution (e.g., C₀t or R₀t analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed).

The words “insertion” and “addition” refer to changes in an amino acidor nucleotide sequence resulting in the addition of one or more aminoacid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment ofSECP which is capable of eliciting an immune response when introducedinto a living organism, for example, a mammal. The term “immunogenicfragment” also includes any polypeptide or oligopeptide fragment of SECPwhich is useful in any of the antibody production methods disclosedherein or known in the art.

The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, or other chemical compounds on asubstrate.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, or other chemical compound having a unique and definedposition on a microarray.

The term “modulate” refers to a change in the activity of SECP. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of SECP.

The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with a second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be in close proximityor contiguous and, where necessary to join two protein coding regions,in the same reading frame.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

“Post-translational modification” of an SECP may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu of SECP.

“Probe” refers to nucleic acid sequences encoding SECP, theircomplements, or fragments thereof, which are used to detect identical,allelic or related nucleic acid sequences. Probes are isolatedoligonucleotides or polynucleotides attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes.

“Primers” are short nucleic acids, usually DNA oligonucleotides, whichmay be annealed to a target polynucleotide by complementarybase-pairing. The primer may then be extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification (and identification) of a nucleic acid sequence, e.g., bythe polymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 25, 30, 40, 50,60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed nucleic acid sequences. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the specification, including the tables, figures, andSequence Listing, may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New YorkN.Y.; Innis, M. et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, San Diego Calif. PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

A “recombinant nucleic acid” is a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook, supra. The term recombinant includes nucleic acids thathave been altered solely by addition, substitution, or deletion of aportion of the nucleic acid. Frequently, a recombinant nucleic acid mayinclude a nucleic acid sequence operably linked to a promoter sequence.Such a recombinant nucleic acid may be part of a vector that is used,for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector, e.g., based on a vaccinia virus, that could be use to vaccinatea mammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

A “regulatory element” refers to a nucleic acid sequence usually derivedfrom untranslated regions of a gene and includes enhancers, promoters,introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elementsinteract with host or viral proteins which control transcription,translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used forlabeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

An “RNA equivalent,” in reference to a DNA sequence, is composed of thesame linear sequence of nucleotides as the reference DNA sequence withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining SECP, nucleic acids encoding SECP, or fragments thereof maycomprise a bodily fluid; an extract from a cell, chromosome, organelle,or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, insolution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, anantagonist, a small molecule, or any natural or synthetic bindingcomposition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least 60% free, preferably at least75% free, and most preferably at least 90% free from other componentswith which they are naturally associated.

A “substitution” refers to the replacement of one or more amino acidresidues or nucleotides by different amino acid residues or nucleotides,respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

A “transcript image” or “expression profile” refers to the collectivepattern of gene expression by a particular cell type or tissue undergiven conditions at a given time.

“Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.The transgenic organisms contemplated in accordance with the presentinvention include bacteria, cyanobacteria, fungi, plants and animals.The isolated DNA of the present invention can be introduced into thehost by methods known in the art, for example infection, transfection,transformation or transconjugation. Techniques for transferring the DNAof the present invention into such organisms are widely known andprovided in references such as Sambrook et al. (1989), supra.

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant. A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternate splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or lack domains that are present in the referencemolecule. Species variants are polynucleotide sequences that vary fromone species to another. The resulting polypeptides will generally havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass “single nucleotide polymorphisms” (SNPs) in which thepolynucleotide sequence varies by one nucleotide base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofpolypeptides may show, for example, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% or greater sequence identity over a certain definedlength of one of the polypeptides.

The Invention

The invention is based on the discovery of new human secreted proteins(SECP), the polynucleotides encoding SECP, and the use of thesecompositions for the diagnosis, treatment, or prevention of cellproliferative, autoimmune/inflammatory, cardiovascular, neurological,and developmental disorders.

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide sequences of the invention. Each polynucleotide and itscorresponding polypeptide are correlated to a single Incyte projectidentification number (Incyte Project ID). Each polypeptide sequence isdenoted by both a polypeptide sequence identification number(Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number(Incyte Polypeptide ID) as shown. Each polynucleotide sequence isdenoted by both a polynucleotide sequence identification number(Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensussequence number (Incyte Polynucleotide ID) as shown.

Table 2 shows sequences with homology to the polypeptides of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database. Columns 1 and 2 show the polypeptide sequenceidentification number (Polypeptide SEQ ID NO:) and the correspondingIncyte polypeptide sequence number (Incyte Polypeptide ID) forpolypeptides of the invention. Column 3 shows the GenBank identificationnumber (GenBank ID NO:) of the nearest GenBank homolog. Column 4 showsthe probability scores for the matches between each polypeptide and itshomolog(s). Column 5 shows the annotation of the GenBank homolog(s)along with relevant citations where applicable, all of which areexpressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of theinvention. Columns 1 and 2 show the polypeptide sequence identificationnumber (SEQ ID NO:) and the corresponding Incyte polypeptide sequencenumber (Incyte Polypeptide ID) for each polypeptide of the invention.Column 3 shows the number of amino acid residues in each polypeptide.Column 4 shows potential phosphorylation sites, and column 5 showspotential glycosylation sites, as determined by the MOTIFS program ofthe GCG sequence analysis software package (Genetics Computer Group,Madison Wis.). Column 6 shows amino acid residues comprising signaturesequences, domains, and motifs. Column 7 shows analytical methods forprotein structure/function analysis and in some cases, searchabledatabases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of theinvention, and these properties establish that the claimed polypeptidesare secreted proteins. For example, SEQ ID NO:1 is 34% identical tohuman seizure related gene 6 (mouse)-like protein, isoform 1 (GenBank IDg6941612) as determined by the Basic Local Alignment Search Tool(BLAST). The BLAST probability score is 8.5e-34, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:1 also contains two CUB domains and a sushi domain(SCR repeat) as determined by searching for statistically significantmatches in the hidden Markov model (HMM)-based PFAM database ofconserved protein family domains. (See Table 3). In an alternativeexample, SEQ ID NO:2 is 40% identical to Drosophila melanogasterperoxidasin precursor (GenBank ID g531385) as determined by the BasicLocal Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 7.8e-266, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:2 also contains a peroxidase domain, four immunoglobulin domains, sixleucine-rich repeats, a leucine-rich repeat C-terminal domain, and a vonWillebrand factor type C domain as determined by searching forstatistically significant matches in the hidden Markov model (HMM)-basedPFAM database of conserved protein family domains. (See Table 3.) Datafrom BLIMPS and MOTIFS analyses provide further corroborative evidencethat SEQ ID NO:2 is a peroxidasin homolog. In an alternative example,SEQ ID NO:4 is 98% identical to Rattus norvegicus neurexophilin (GenBankID g508574) as determined by the Basic Local Alignment Search Tool(BLAST). (See Table 2.) The BLAST probability score is 4.7e-148, whichindicates the probability of obtaining the observed polypeptide sequencealignment by chance. Data from SPSCAN and BLAST_PRODOM analyses providefurther corroborative evidence that SEQ ID NO:4 is a secretedneurexophilin. In an alternative example, SEQ ID NO:6 is 68% identicalto pig preprosecretin (GenBank ID g164671) as determined by the BasicLocal Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 2.3e-36, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:6 has a signal peptide, as predicted by HMMER and SPSCAN. SEQ ID NO:6also contains a polypeptide hormone domain as determined by searchingfor statistically significant matches in the hidden Markov model(HMM)-based PFAM database of conserved protein family domains. (SeeTable 3.) The presence of this domain is confirmed by BLIMPS and MOTIFSanalyses, providing further corroborative evidence that SEQ ID NO:6 is asecreted hormone. In an alternative example, SEQ ID NO:28 is 78%identical to Mus musculus nodal, a TGF-β like gene (GenBank ID g296605)as determined by the Basic Local Alignment Search Tool (BLAST). (SeeTable 2.) The BLAST probability score is 7.5e-148, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:28 also contains a TGF-β like domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM database of conserved protein family domains.(See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analysesprovide further corroborative evidence that SEQ ID NO:28 is a TGF-β likeprotein. In an alternative example, SEQ ID NO:63 is 86% identical to ratlate gestation lung protein 1 (GenBank ID g4324682) as determined by theBasic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 3.4e-97, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:63 also contains an SCP (sperm-coating glycoprotein)-likeextracellular protein domain as determined by searching forstatistically significant matches in the hidden Markov model (HMM)-basedPFAM database of conserved protein family domains. (See Table 3.) Datafrom BLIMPS and MOTIFS analyses provide further corroborative evidencethat SEQ ID NO:63 is a protease inhibitor-like protein. SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7-27, and SEQ ID NO:29-62 were analyzed and annotatedin a similar manner. The algorithms and parameters for the analysis ofSEQ ID NO:1-63 are described in Table 7.

As shown in Table 4, the full length polynucleotide sequences of thepresent invention were assembled using cDNA sequences or coding (exon)sequences derived from genomic DNA, or any combination of these twotypes of sequences. Columns 1 and 2 list the polynucleotide sequenceidentification number (Polynucleotide SEQ ID NO:) and the correspondingIncyte polynucleotide consensus sequence number (Incyte PolynucleotideID) for each polynucleotide of the invention. Column 3 shows the lengthof each polynucleotide sequence in basepairs. Column 4 lists fragmentsof the polynucleotide sequences which are useful, for example, inhybridization or amplification technologies that identify SEQ IDNO:64-126 or that distinguish between SEQ ID NO:64-126 and relatedpolynucleotide sequences. Column 5 shows identification numberscorresponding to cDNA sequences, coding sequences (exons) predicted fromgenomic DNA, and/or sequence assemblages comprised of both cDNA andgenomic DNA. These sequences were used to assemble the full lengthpolynucleotide sequences of the invention. Columns 6 and 7 of Table 4show the nucleotide start (5′) and stop (3′) positions of the cDNAand/or genomic sequences in column 5 relative to their respective fulllength sequences.

The identification numbers in Column 5 of Table 4 may referspecifically, for example, to Incyte cDNAs along with theircorresponding cDNA libraries. For example, 2719959T6 is theidentification number of an Incyte cDNA sequence, and LUNGTUT10 is thecDNA library from which it is derived. Incyte cDNAs for which cDNAlibraries are not indicated were derived from pooled cDNA libraries(e.g., 56002879J1). Alternatively, the identification numbers in column5 may refer to GenBank cDNAs or ESTs (e.g., g1547765) which contributedto the assembly of the full length polynucleotide sequences. Inaddition, the identification numbers in column 5 may identify sequencesderived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database(i.e., those sequences including the designation “ENST”). Alternatively,the identification numbers in column 5 may be derived from the NCBIRefSeq Nucleotide Sequence Records Database (i.e., those sequencesincluding the designation “NM” or “NT”) or the NCBI RefSeq ProteinSequence Records (i.e., those sequences including the designation “NP”).Alternatively, the identification numbers in column 5 may refer toassemblages of both cDNA and Genscan-predicted exons brought together byan “exon stitching” algorithm. For example,FL_XXXXXX_N_(1—)N_(2—)YYYYY_N_(3—)N₄ represents a “stitched” sequence inwhich XXXXXX is the identification number of the cluster of sequences towhich the algorithm was applied, and YYYYY is the number of theprediction generated by the algorithm, and N_(1, 2, 3) . . . , ifpresent, represent specific exons that may have been manually editedduring analysis (See Example V). Alternatively, the identificationnumbers in column 5 may refer to assemblages of exons brought togetherby an “exon-stretching” algorithm. For example,FLXXXXXX_gAAAAA_gBBBBB_(—)1_N is the identification number of a“stretched” sequence, with XXXXXX being the Incyte projectidentification number, gAAAAA being the GenBank identification number ofthe human genomic sequence to which the “exon-stretching” algorithm wasapplied, gBBBBB being the GenBank identification number or NCBI RefSeqidentification number of the nearest GenBank protein homolog, and Nreferring to specific exons (See Example V). In instances where a RefSeqsequence was used as a protein homolog for the “exon-stretching”algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) may beused in place of the GenBank identifier (i.e., gBBBBB).

Alternatively, a prefix identifies component sequences that werehand-edited, predicted from genomic DNA sequences, or derived from acombination of sequence analysis methods. The following Table listsexamples of component sequence prefixes and corresponding sequenceanalysis methods associated with the prefixes (see Example IV andExample V).

Prefix Type of analysis and/or examples of programs GNN, GFG, Exonprediction from genomic sequences using, for example, ENST GENSCAN(Stanford University, CA, USA) or FGENES (Computer Genomics Group, TheSanger Centre, Cambridge, UK). GBI Hand-edited analysis of genomicsequences. FL Stitched or stretched genomic sequences (see Example V).INCY Full length transcript and exon prediction from mapping of ESTsequences to the genome. Genomic location and EST composition data arecombined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverageshown in column 5 was obtained to confirm the final consensuspolynucleotide sequence, but the relevant Incyte cDNA identificationnumbers are not shown.

Table 5 shows the representative cDNA libraries for those full lengthpolynucleotide sequences which were assembled using Incyte cDNAsequences. The representative cDNA library is the Incyte cDNA librarywhich is most frequently represented by the Incyte cDNA sequences whichwere used to assemble and confirm the above polynucleotide sequences.The tissues and vectors which were used to construct the cDNA librariesshown in Table 5 are described in Table 6.

The invention also encompasses SECP variants. A preferred SECP variantis one which has at least about 80%, or alternatively at least about90%, or even at least about 95% amino acid sequence identity to the SECPamino acid sequence, and which contains at least one functional orstructural characteristic of SECP.

The invention also encompasses polynucleotides which encode SECP. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:64-126, which encodes SECP. The polynucleotide sequences of SEQ IDNO:64-126, as presented in the Sequence Listing, embrace the equivalentRNA sequences, wherein occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The invention also encompasses a variant of a polynucleotide sequenceencoding SECP. In particular, such a variant polynucleotide sequencewill have at least about 70%, or alternatively at least about 85%, oreven at least about 95% polynucleotide sequence identity to thepolynucleotide sequence encoding SECP. A particular aspect of theinvention encompasses a variant of a polynucleotide sequence comprisinga sequence selected from the group consisting of SEQ ID NO:64-126 whichhas at least about 70%, or alternatively at least about 85%, or even atleast about 95% polynucleotide sequence identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO:64-126. Any oneof the polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of SECP.

In addition, or in the alternative, a polynucleotide variant of theinvention is a splice variant of a polynucleotide sequence encodingSECP. A splice variant may have portions which have significant sequenceidentity to the polynucleotide sequence encoding SECP, but willgenerally have a greater or lesser number of polynucleotides due toadditions or deletions of blocks of sequence arising from alternatesplicing of exons during mRNA processing. A splice variant may have lessthan about 70%, or alternatively less than about 60%, or alternativelyless than about 50% polynucleotide sequence identity to thepolynucleotide sequence encoding SECP over its entire length; however,portions of the splice variant will have at least about 70%, oralternatively at least about 85%, or alternatively at least about 95%,or alternatively 100% polynucleotide sequence identity to portions ofthe polynucleotide sequence encoding SECP. Any one of the splicevariants described above can encode an amino acid sequence whichcontains at least one functional or structural characteristic of SECP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding SECP, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring SECP, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode SECP and its variants aregenerally capable of hybridizing to the nucleotide sequence of thenaturally occurring SECP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding SECP or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding SECP and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeSECP and SECP derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding SECP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:64-126 and fragments thereofunder various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.) Hybridization conditions, includingannealing and wash conditions, are described in “Definitions.”

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems),thermostable T7 polymerase (Amersham Pharmacia Biotech, PiscatawayN.J.), or combinations of polymerases and proofreading exonucleases suchas those found in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the MICROLAB 2200 liquid transfer system (Hamilton,Reno Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABICATALYST 800 thermal cycler (Applied Biosystems). Sequencing is thencarried out using either the ABI 373 or 377 DNA sequencing system(Applied. Biosystems), the MEGABACE 1000 DNA sequencing system(Molecular Dynamics, Sunnyvale Calif.), or other systems known in theart. The resulting sequences are analyzed using a variety of algorithmswhich are well known in the art. (See, e.g., Ausubel, F. M. (1997) ShortProtocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit7.7; Meyers, R. A. (1995) Molecular Biology and Biotechnology, WileyVCH, New York N.Y., pp. 856-853.)

The nucleic acid sequences encoding SECP may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppliedBiosystems), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode SECP may be cloned in recombinant DNAmolecules that direct expression of SECP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express SECP.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter SECP-encodingsequences for a variety of purposes including, but not limited to,modification of the cloning, processing, and/or expression of the geneproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides may be used to engineerthe nucleotide sequences. For example, oligonucleotide-mediatedsite-directed mutagenesis may be used to introduce mutations that createnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of SECP, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

In another embodiment, sequences encoding SECP may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223;and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.)Alternatively, SECP itself or a fragment thereof may be synthesizedusing chemical methods. For example, peptide synthesis can be performedusing various solution-phase or solid-phase techniques. (See, e.g.,Creighton, T. (1984) Proteins, Structures and Molecular Properties, WHFreeman, New York N.Y., pp. 55-60; and Roberge, J. Y. et al. (1995)Science 269:202-204.) Automated synthesis may be achieved using the ABI431A peptide synthesizer (Applied Biosystems). Additionally, the aminoacid sequence of SECP, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide or a polypeptide having asequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g., Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, supra, pp. 28-53.)

In order to express a biologically active SECP, the nucleotide sequencesencoding SECP or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesequences encoding SECP. Such elements may vary in their strength andspecificity. Specific initiation signals may also be used to achievemore efficient translation of sequences encoding SECP. Such signalsinclude the ATG initiation codon and adjacent sequences, e.g. the Kozaksequence. In cases where sequences encoding SECP and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding SECP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding SECP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See,e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; TheMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.Sci. USA 81:3655-3659; and Harrington, J. J. et al. (1997) Nat. Genet.15:345-355.) Expression vectors derived from retroviruses, adenoviruses,or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for delivery of nucleotide sequences to the targeted organ,tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998)Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad.Sci. USA 90 (13):6340-6344; Buller, R. M. et al. (1985) Nature317(6040):813-815; McGregor, D. P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, I. M. and N. Somia (1997) Nature 389:239-242.)The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding SECP. For example, routine cloning, subcloning, and propagationof polynucleotide sequences encoding SECP can be achieved using amultifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JollaCalif.) or PSPORT1 plasmid (Life Technologies). Ligation of sequencesencoding SECP into the vector's multiple cloning site disrupts the lacZgene, allowing a colorimetric screening procedure for identification oftransformed bacteria containing recombinant molecules. In addition,these vectors may be useful for in vitro transcription, dideoxysequencing, single strand rescue with helper phage, and creation ofnested deletions in the cloned sequence. (See, e.g., Van Heeke, G. andS. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of SECP are needed, e.g. for the production of antibodies,vectors which direct high level expression of SECP may be used. Forexample, vectors containing the strong, inducible SP6 or T7bacteriophage promoter may be used.

Yeast expression systems may be used for production of SECP. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, 1995, supra; Bitter,G. A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C. A. etal. (1994) Bio/Technology 12:181-184.)

Plant systems may also be used for expression of SECP. Transcription ofsequences encoding SECP may be driven by viral promoters, e.g., the 35Sand 19S promoters of CaMV used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., The McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New YorkN.Y., pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding SECP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses SECP in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. etal. (1997) Nat. Genet. 15:345-355.)

For long term production of recombinant proteins in mammalian systems,stable expression of SECP in cell lines is preferred. For example,sequences encoding SECP can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ and apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin,F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable geneshave been described, e.g., trpB and hisD, which alter cellularrequirements for metabolites. (See, e.g., Hartman, S. C. and R. C.Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.) Visiblemarkers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),β glucuronidase and its substrate β-glucuronide, or luciferase and itssubstrate luciferin may be used. These markers can be used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system.(See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingSECP is inserted within a marker gene sequence, transformed cellscontaining sequences encoding SECP can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding SECP under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingSECP and that express SECP may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification,and protein bioassay or immunoassay techniques which include membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of SECPusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on SECPis preferred, but a competitive binding assay may be employed. These andother assays are well known in the art. (See, e.g., Hampton, R. et al.(1990) Serological Methods, a Laboratory Manual, APS Press, St. PaulMinn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols inImmunology, Greene Pub. Associates and Wiley-Interscience, New YorkN.Y.; and Pound, J. D. (1998) Immunochemical Protocols, Humana Press,Totowa N.J.)

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding SECP includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding SECP,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitablereporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding SECP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeSECP may be designed to contain signal sequences which direct secretionof SECP through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding SECP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric SECPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of SECP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the SECP encodingsequence and the heterologous protein sequence, so that SECP may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch. 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

In a further embodiment of the invention, synthesis of radiolabeled SECPmay be achieved in vitro using the TNT rabbit reticulocyte lysate orwheat germ extract system (Promega). These systems couple transcriptionand translation of protein-coding sequences operably associated with theT7, T3, or SP6 promoters. Translation takes place in the presence of aradiolabeled amino acid precursor, for example, ³⁵S-methionine.

SECP of the present invention or fragments thereof may be used to screenfor compounds that specifically bind to SECP. At least one and up to aplurality of test compounds may be screened for specific binding toSECP. Examples of test compounds include antibodies, oligonucleotides,proteins (e.g., receptors), or small molecules.

In one embodiment, the compound thus identified is closely related tothe natural ligand of SECP, e.g., a ligand or fragment thereof, anatural substrate, a structural or functional mimetic, or a naturalbinding partner. (See, e.g., Coligan, J. E. et al. (1991) CurrentProtocols in Immunology 1 (2): Chapter 5.) Similarly, the compound canbe closely related to the natural receptor to which SECP binds, or to atleast a fragment of the receptor, e.g., the ligand binding site. Ineither case, the compound can be rationally designed using knowntechniques. In one embodiment, screening for these compounds involvesproducing appropriate cells which express SECP, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing SECP or cellmembrane fractions which contain SECP are then contacted with a testcompound and binding, stimulation, or inhibition of activity of eitherSECP or the compound is analyzed.

An assay may simply test binding of a test compound to the polypeptide,wherein binding is detected by a fluorophore, radioisotope, enzymeconjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with SECP,either in solution or affixed to a solid support, and detecting thebinding of SECP to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

SECP of the present invention or fragments thereof may be used to screenfor compounds that modulate the activity of SECP. Such compounds mayinclude agonists, antagonists, or partial or inverse agonists. In oneembodiment, an assay is performed under conditions permissive for SECPactivity, wherein SECP is combined with at least one test compound, andthe activity of SECP in the presence of a test compound is compared withthe activity of SECP in the absence of the test compound. A change inthe activity of SECP in the presence of the test compound is indicativeof a compound that modulates the activity of SECP. Alternatively, a testcompound is combined with an in vitro or cell-free system comprisingSECP under conditions suitable for SECP activity, and the assay isperformed. In either of these assays, a test compound which modulatesthe activity of SECP may do so indirectly and need not come in directcontact with the test compound. At least one and up to a plurality oftest compounds may be screened.

In another embodiment, polynucleotides encoding SECP or their mammalianhomologs may be “knocked out” in an animal model system using homologousrecombination in embryonic stem (ES) cells. Such techniques are wellknown in the art and are useful for the generation of animal models ofhuman disease. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No.5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cellline, are derived from the early mouse embryo and grown in culture. TheES cells are transformed with a vector containing the gene of interestdisrupted by a marker gene, e.g., the neomycin phosphotransferase gene(neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vectorintegrates into the corresponding region of the host genome byhomologous recombination. Alternatively, homologous recombination takesplace using the Cre-loxP system to knockout a gene of interest in atissue- or developmental stage-specific manner (Marth, J. D. (1996)Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic AcidsRes. 25:4323-4330). Transformed ES cells are identified andmicroinjected into mouse cell blastocysts such as those from the C57BL/6mouse strain. The blastocysts are surgically transferred topseudopregnant dams, and the resulting chimeric progeny are genotypedand bred to produce heterozygous or homozygous strains. Transgenicanimals thus generated may be tested with potential therapeutic or toxicagents.

Polynucleotides encoding SECP may also be manipulated in vitro in EScells derived from human blastocysts. Human ES cells have the potentialto differentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding SECP can also be used to create “knockin”humanized animals (pigs) or transgenic animals (mice or rats) to modelhuman disease. With knockin technology, a region of a polynucleotideencoding SECP is injected into animal ES cells, and the injectedsequence integrates into the animal cell genome. Transformed cells areinjected into blastulae, and the blastulae are implanted as describedabove. Transgenic progeny or inbred lines are studied and treated withpotential pharmaceutical agents to obtain information on treatment of ahuman disease. Alternatively, a mammal inbred to overexpress SECP, e.g.,by secreting SECP in its milk, may also serve as a convenient source ofthat protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of SECP and secreted proteins. Inaddition, the expression of SECP is closely associated with normal andtumorous lung, heart, brain, skin, colon epithelium, and cardiovasculartissues, as well as, neurological, urinary, reproductive, digestive,immunological, diseased, and tumorous tissues. Therefore, SECP appearsto play a role in cell proliferative, autoimmune/inflammatory,cardiovascular, neurological, and developmental disorders. In thetreatment of disorders associated with increased SECP expression oractivity, it is desirable to decrease the expression or activity ofSECP. In the treatment of disorders associated with decreased SECPexpression or activity, it is desirable to increase the expression oractivity of SECP.

Therefore, in one embodiment, SECP or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of SECP. Examples ofsuch disorders include, but are not limited to, a cell proliferativedisorder such as actinic keratosis, arteriosclerosis, atherosclerosis,bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, a cancer of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatorydisorder such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma; acardiovascular disorder such as congestive heart failure, ischemic heartdisease, angina pectoris, myocardial infarction, hypertensive heartdisease, degenerative valvular heart disease, calcific aortic valvestenosis, congenitally bicuspid aortic valve, mitral annularcalcification, mitral valve prolapse, rheumatic fever and rheumaticheart disease, infective endocarditis, nonbacterial thromboticendocarditis, endocarditis of systemic lupus erythematosus, carcinoidheart disease, cardiomyopathy, myocarditis, pericarditis, neoplasticheart disease, congenital heart disease, complications of cardiactransplantation, arteriovenous fistula, atherosclerosis, hypertension,vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicoseveins, thrombophlebitis and phlebothrombosis, vascular tumors, andcomplications of thrombolysis, balloon angioplasty, vascularreplacement, and coronary artery bypass graft surgery; a neurologicaldisorder such as epilepsy, ischemic cerebrovascular disease, stroke,cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington'sdisease, dementia, Parkinson's disease and other extrapyramidaldisorders, amyotrophic lateral sclerosis and other motor neurondisorders, progressive neural muscular atrophy, retinitis pigmentosa,hereditary ataxias, multiple sclerosis and other demyelinating diseases,bacterial and viral meningitis, brain abscess, subdural empyema,epidural abscess, suppurative intracranial thrombophlebitis, myelitisand radiculitis, viral central nervous system disease, prion diseasesincluding kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; and a developmental disorder such asrenal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinaryabnormalities, and mental retardation), Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss.

In another embodiment, a vector capable of expressing SECP or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofSECP including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantiallypurified SECP in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of SECP including, but not limitedto, those provided above.

In still another embodiment, an agonist which modulates the activity ofSECP may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of SECP including, butnot limited to, those listed above.

In a further embodiment, an antagonist of SECP may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of SECP. Examples of such disorders include, butare not limited to, those cell proliferative, autoimmune/inflammatory,cardiovascular, neurological, and developmental disorders describedabove. In one aspect, an antibody which specifically binds SECP may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissues whichexpress SECP.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding SECP may be administered to a subject to treator prevent a disorder associated with increased expression or activityof SECP including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of SECP may be produced using methods which are generallyknown in the art In particular, purified SECP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind SECP. Antibodies to SECP may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are generally preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith SECP or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to SECP have an amino acid sequence consisting of atleast about 5 amino acids, and generally will consist of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein. Short stretches of SECP amino acids maybe fused with those of another protein, such as KLH, and antibodies tothe chimeric molecule may be produced.

Monoclonal antibodies to SECP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S. P. etal. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce SECP-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for SECP mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between SECP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering SECP epitopes is generally used, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for SECP. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of SECP-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple SECP epitopes, represents the average affinity,or avidity, of the antibodies for SECP. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular SECP epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theSECP-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of SECP, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. and A.Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &Sons, New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is generally employed in proceduresrequiring precipitation of SECP-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingSECP, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, modifications of gene expression can beachieved by designing complementary sequences or antisense molecules(DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding SECP. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding SECP. (See, e.g., Agrawal, S., ed. (1996)Antisense Therapeutics, Humana Press Inc., Totawa N.J.)

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein. (See, e.g., Slater, J. E. et al. (1998) J. AllergyClin. Immunol. 102 (3):469-475; and Scanlon, K. J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introducedintracellularly through the use of viral vectors, such as retrovirus andadeno-associated virus vectors. (See, e.g., Miller, A. D. (1990) Blood76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol.Ther. 63 (3):323-347.) Other gene delivery mechanisms includeliposome-derived systems, artificial viral envelopes, and other systemsknown in the art. (See, e.g., Rossi, J. J. (1995) Br. Med. Bull. 51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids Res.25(14):2730-2736.)

In another embodiment of the invention, polynucleotides encoding SECPmay be used for somatic or germline gene therapy. Gene therapy may beperformed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669-672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475-480; Bordignon, C. et al. (1995) Science270:470475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HIV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus(HBV, HCV); fungal parasites, such as Candida albicans andParacoccidioides brasiliensis; and protozoan parasites such asPlasmodium falciparum and Trypanosoma cruzi). In the case where agenetic deficiency in SECP expression or regulation causes disease, theexpression of SECP from an appropriate population of transduced cellsmay alleviate the clinical manifestations caused by the geneticdeficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in SECP are treated by constructing mammalian expressionvectors encoding SECP and introducing these vectors by mechanical meansinto SECP-deficient cells. Mechanical transfer technologies for use withcells in vivo or ex vitro include (i) direct DNA microinjection intoindividual cells, (ii) ballistic gold particle delivery, (iii)liposome-mediated transfection, (iv) receptor-mediated gene transfer,and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson(1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510;Boulay, J-L. and H. Rećipon (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of SECPinclude, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT,PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF,PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). SECPmay be expressed using (i) a constitutively active promoter, (e.g., fromcytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidinekinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., thetetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin.Biotechnol. 9:451-456), commercially available in the T-REX plasmid(Invitrogen)); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and H. M. Blau, supra)), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding SECP from a normalindividual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and A. J. Eb (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to SECP expression are treated byconstructing a retrovirus vector consisting of (i) the polynucleotideencoding SECP under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 toRigg (“Method for obtaining retrovirus packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4⁺ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)Blood 89:2283-2290).

In the alternative, an adenovirus-based gene therapy delivery system isused to deliver polynucleotides encoding SECP to cells which have one ormore genetic abnormalities with respect to the expression of SECP. Theconstruction and packaging of adenovirus-based vectors are well known tothose with ordinary skill in the art. Replication defective adenovirusvectors have proven to be versatile for importing genes encodingimmunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially usefuladenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano(“Adenovirus vectors for gene therapy”), hereby incorporated byreference. For adenoviral vectors, see also Antinozzi, P. A. et al.(1999) Annu. Rev. Nutr. 19:511-544 and Verma, I. M. and N. Somia (1997)Nature 18:389:239-242, both incorporated by reference herein.

In another alternative, a herpes-based, gene therapy delivery system isused to deliver polynucleotides encoding SECP to target cells which haveone or more genetic abnormalities with respect to the expression ofSECP. The use of herpes simplex virus (HSV)-based vectors may beespecially valuable for introducing SECP to cells of the central nervoussystem, for which HSV has a tropism. The construction and packaging ofherpes-based vectors are well known to those with ordinary skill in theart. A replication-competent herpes simplex virus (HSV) type 1-basedvector has been used to deliver a reporter gene to the eyes of primates(Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The construction of aHSV-1 virus vector has also been disclosed in detail in U.S. Pat. No.5,804,413 to DeLuca (“Herpes simplex virus strains for gene transfer”),which is hereby incorporated by reference. U.S. Pat. No. 5,804,413teaches the use of recombinant HSV d92 which consists of a genomecontaining at least one exogenous gene to be transferred to a cell underthe control of the appropriate promoter for purposes including humangene therapy. Also taught by this patent are the construction and use ofrecombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSVvectors, see also Goins, W. F. et al. (1999) J. Virol. 73:519-532 andXu, H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated byreference. The manipulation of cloned herpesvirus sequences, thegeneration of recombinant virus following the transfection of multipleplasmids containing different segments of the large herpesvirus genomes,the growth and propagation of herpesvirus, and the infection of cellswith herpesvirus are techniques well known to those of ordinary skill inthe art.

In another alternative, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver polynucleotides encoding SECP to targetcells. The biology of the prototypic alphavirus, Semliki Forest Virus(SFV), has been studied extensively and gene transfer vectors have beenbased on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genomicRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for SECP into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of SECP-coding RNAs and the synthesis of high levels ofSECP in vector transduced cells. While alphavirus infection is typicallyassociated with cell lysis within a few days, the ability to establish apersistent infection in hamster normal kidney cells (BHK-21) with avariant of Sindbis virus (SIN) indicates that the lytic replication ofalphaviruses can be altered to suit the needs of the gene therapyapplication (Dryga, S. A. et al. (1997) Virology 228:74-83). The widehost range of alphaviruses will allow the introduction of SECP into avariety of cell types. The specific transduction of a subset of cells ina population may require the sorting of cells prior to transduction. Themethods of manipulating infectious cDNA clones of alphaviruses,performing alphavirus cDNA and RNA transfections, and performingalphavirus infections, are well known to those with ordinary skill inthe art.

Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, may also beemployed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature. (See,e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecularand Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp.163-177.) A complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingSECP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding SECP. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

An additional embodiment of the invention encompasses a method forscreening for a compound which is effective in altering expression of apolynucleotide encoding SECP. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased SECPexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding SECP may be therapeuticallyuseful, and in the treatment of disorders associated with decreased SECPexpression or activity, a compound which specifically promotesexpression of the polynucleotide encoding SECP may be therapeuticallyuseful.

At least one, and up to a plurality, of test compounds may be screenedfor effectiveness in altering expression of a specific polynucleotide. Atest compound may be obtained by any method commonly known in the art,including chemical modification of a compound known to be effective inaltering polynucleotide expression; selection from an existing,commercially-available or proprietary library of naturally-occurring ornon-natural chemical compounds; rational design of a compound based onchemical and/or structural properties of the target polynucleotide; andselection from a library of chemical compounds created combinatoriallyor randomly. A sample comprising a polynucleotide encoding SECP isexposed to at least one test compound thus obtained. The sample maycomprise, for example, an intact or permeabilized cell, or an in vitrocell-free or reconstituted biochemical system. Alterations in theexpression of a polynucleotide encoding SECP are assayed by any methodcommonly known in the art. Typically, the expression of a specificnucleotide is detected by hybridization with a probe having a nucleotidesequence complementary to the sequence of the polynucleotide encodingSECP. The amount of hybridization may be quantified, thus forming thebasis for a comparison of the expression of the polynucleotide both withand without exposure to one or more test compounds. Detection of achange in the expression of a polynucleotide exposed to a test compoundindicates that the test compound is effective in altering the expressionof the polynucleotide. A screen for a compound effective in alteringexpression of a specific polynucleotide can be carried out, for example,using a Schizosaccharomyces pombe gene expression system (Atkins, D. etal. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) NucleicAcids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L.et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) Nat.Biotechnol. 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such ashumans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administrationof a composition which generally comprises an active ingredientformulated with a pharmaceutically acceptable excipient. Excipients mayinclude, for example, sugars, starches, celluloses, gums, and proteins.Various formulations are commonly known and are thoroughly discussed inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.). Such compositions may consist of SECP,antibodies to SECP, and mimetics, agonists, antagonists, or inhibitorsof SECP.

The compositions utilized in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid ordry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thecase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery has the advantage of administrationwithout needle injection, and obviates the need for potentially toxicpenetration enhancers.

Compositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for directintracellular delivery of macromolecules comprising SECP or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, SECP or a fragment thereofmay be joined to a short cationic N-terminal portion from the HIV Tat-1protein. Fusion proteins thus generated have been found to transduceinto the cells of all tissues, including the brain, in a mouse modelsystem (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example SECP or fragments thereof, antibodies of SECP,and agonists, antagonists or inhibitors of SECP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies are used to formulate a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that includes the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind SECP may beused for the diagnosis of disorders characterized by expression of SECP,or in assays to monitor patients being treated with SECP or agonists,antagonists, or inhibitors of SECP. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for SECP include methods which utilizethe antibody and a label to detect SECP in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring SECP, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of SECP expression. Normal or standard values for SECPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, for example, human subjects, withantibodies to SECP under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, such as photometric means. Quantities of SECP expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingSECP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantify gene expression in biopsied tissues in which expression of SECPmay be correlated with disease. The diagnostic assay may be used todetermine absence, presence, and excess expression of SECP, and tomonitor regulation of SECP levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding SECP or closely related molecules may be used to identifynucleic acid sequences which encode SECP. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification willdetermine whether the probe identifies only naturally occurringsequences encoding SECP, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, and mayhave at least 50% sequence identity to any of the SECP encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:64-126 or fromgenomic sequences including promoters, enhancers, and introns of theSECP gene.

Means for producing specific hybridization probes for DNAs encoding SECPinclude the cloning of polynucleotide sequences encoding SECP or SECPderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding SECP may be used for the diagnosis ofdisorders associated with expression of SECP. Examples of such disordersinclude, but are not limited to, a cell proliferative disorder such asactinic keratosis, arteriosclerosis, atherosclerosis, bursitis,cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, a cancer of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatorydisorder such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma; acardiovascular disorder such as congestive heart failure, ischemic heartdisease, angina pectoris, myocardial infarction, hypertensive heartdisease, degenerative valvular heart disease, calcific aortic valvestenosis, congenitally bicuspid aortic valve, mitral annularcalcification, mitral valve prolapse, rheumatic fever and rheumaticheart disease, infective endocarditis, nonbacterial thromboticendocarditis, endocarditis of systemic lupus erythematosus, carcinoidheart disease, cardiomyopathy, myocarditis, pericarditis, neoplasticheart disease, congenital heart disease, complications of cardiactransplantation, arteriovenous fistula, atherosclerosis, hypertension,vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicoseveins, thrombophlebitis and phlebothrombosis, vascular tumors, andcomplications of thrombolysis, balloon angioplasty, vascularreplacement, and coronary artery bypass graft surgery; a neurologicaldisorder such as epilepsy, ischemic cerebrovascular disease, stroke,cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington'sdisease, dementia, Parkinson's disease and other extrapyramidaldisorders, amyotrophic lateral sclerosis and other motor neurondisorders, progressive neural muscular atrophy, retinitis pigmentosa,hereditary ataxias, multiple sclerosis and other demyelinating diseases,bacterial and viral meningitis, brain abscess, subdural empyema,epidural abscess, suppurative intracranial thrombophlebitis, myelitisand radiculitis, viral central nervous system disease, prion diseasesincluding kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; and a developmental disorder such asrenal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinaryabnormalities, and mental retardation), Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss. The polynucleotidesequences encoding SECP may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and multiformat ELISA-like assays; and in microarraysutilizing fluids or tissues from patients to detect altered SECPexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding SECP may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingSECP may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantified and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding SECP in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of SECP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding SECP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding SECP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding SECP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding SECP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived from thepolynucleotide sequences encoding SECP may be used to detect singlenucleotide polymorphisms (SNPs). SNPs are substitutions, insertions anddeletions that are a frequent cause of inherited or acquired geneticdisease in humans. Methods of SNP detection include, but are not limitedto, single-stranded conformation polymorphism (SSCP) and fluorescentSSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from thepolynucleotide sequences encoding SECP are used to amplify DNA using thepolymerase chain reaction (PCR). The DNA may be derived, for example,from diseased or normal tissue, biopsy samples, bodily fluids, and thelike. SNPs in the DNA cause differences in the secondary and tertiarystructures of PCR products in single-stranded form, and thesedifferences are detectable using gel electrophoresis in non-denaturinggels. In fSCCP, the oligonucleotide primers are fluorescently labeled,which allows detection of the amplimers in high-throughput equipmentsuch as DNA sequencing machines. Additionally, sequence databaseanalysis methods, termed in silico SNP (is SNP), are capable ofidentifying polymorphisms by comparing the sequence of individualoverlapping DNA fragments which assemble into a common consensussequence. These computer-based methods filter out sequence variationsdue to laboratory preparation of DNA and sequencing errors usingstatistical models and automated analyses of DNA sequence chromatograms.In the alternative, SNPs may be detected and characterized by massspectrometry using, for example, the high throughput MASSARRAY system(Sequenom, Inc., San Diego Calif.).

Methods which may also be used to quantify the expression of SECPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin a high-throughput format where the oligomer or polynucleotide ofinterest is presented in various dilutions and a spectrophotometric orcolorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used aselements on a microarray. The microarray can be used in transcriptimaging techniques which monitor the relative expression levels of largenumbers of genes simultaneously as described below. The microarray mayalso be used to identify genetic variants, mutations, and polymorphisms.This information may be used to determine gene function, to understandthe genetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

In another embodiment, SECP, fragments of SECP, or antibodies specificfor SECP may be used as elements on a microarray. The microarray may beused to monitor or measure protein-protein interactions, drug-targetinteractions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of thepresent invention to generate a transcript image of a tissue or celltype. A transcript image represents the global pattern of geneexpression by a particular tissue or cell type. Global gene expressionpatterns are analyzed by quantifying the number of expressed genes andtheir relative abundance under given conditions and at a given time.(See Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat.No. 5,840,484, expressly incorporated by reference herein.) Thus atranscript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totality oftranscripts or reverse transcripts of a particular tissue or cell type.In one embodiment, the hybridization takes place in high-throughputformat, wherein the polynucleotides of the present invention or theircomplements comprise a subset of a plurality of elements on amicroarray. The resultant transcript image would provide a profile ofgene activity.

Transcript images may be generated using transcripts isolated fromtissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo, as in thecase of a tissue or biopsy sample, or in vitro, as in the case of a cellline.

Transcript images which profile the expression of the polynucleotides ofthe present invention may also be used in conjunction with in vitromodel systems and preclinical evaluation of pharmaceuticals, as well astoxicological testing of industrial and naturally-occurringenvironmental compounds. All compounds induce characteristic geneexpression patterns, frequently termed molecular fingerprints ortoxicant signatures, which are indicative of mechanisms of action andtoxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159;Steiner, S, and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471,expressly incorporated by reference herein). If a test compound has asignature similar to that of a compound with known toxicity, it islikely to share those toxic properties. These fingerprints or signaturesare most useful and refined when they contain expression informationfrom a large number of genes and gene families. Ideally, a genome-widemeasurement of expression provides the highest quality signature. Evengenes whose expression is not altered by any tested compounds areimportant as well, as the levels of expression of these genes are usedto normalize the rest of the expression data. The normalizationprocedure is useful for comparison of expression data after treatmentwith different compounds. While the assignment of gene function toelements of a toxicant signature aids in interpretation of toxicitymechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction oftoxicity. (See, for example, Press Release 00-02 from the NationalInstitute of Environmental Health Sciences, released Feb. 29, 2000,available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore,it is important and desirable in toxicological screening using toxicantsignatures to include all expressed gene sequences.

In one embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another particular embodiment relates to the use of the polypeptidesequences of the present invention to analyze the proteome of a tissueor cell type. The term proteome refers to the global pattern of proteinexpression in a particular tissue or cell type. Each protein componentof a proteome can be subjected individually to further analysis.Proteome expression patterns, or profiles, are analyzed by quantifyingthe number of expressed proteins and their relative abundance undergiven conditions and at a given time. A profile of a cell's proteome maythus be generated by separating and analyzing the polypeptides of aparticular tissue or cell type. In one embodiment, the separation isachieved using two-dimensional gel electrophoresis, in which proteinsfrom a sample are separated by isoelectric focusing in the firstdimension, and then according to molecular weight by sodium dodecylsulfate slab gel electrophoresis in the second dimension (Steiner andAnderson, supra). The proteins are visualized in the gel as discrete anduniquely positioned spots, typically by staining the gel with an agentsuch as Coomassie Blue or silver or fluorescent stains. The opticaldensity of each protein spot is generally proportional to the level ofthe protein in the sample. The optical densities of equivalentlypositioned protein spots from different samples, for example, frombiological samples either treated or untreated with a test compound ortherapeutic agent, are compared to identify any changes in protein spotdensity related to the treatment. The proteins in the spots arepartially sequenced using, for example, standard methods employingchemical or enzymatic cleavage followed by mass spectrometry. Theidentity of the protein in a spot may be determined by comparing itspartial sequence, preferably of at least 5 contiguous amino acidresidues, to the polypeptide sequences of the present invention. In somecases, further sequence data may be obtained for definitive proteinidentification.

A proteomic profile may also be generated using antibodies specific forSECP to quantify the levels of SECP expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by exposing the microarray to the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999)Biotechniques 27:778-788). Detection may be performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the polypeptides of the presentinvention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the polypeptides of the present invention. The amount ofprotein recognized by the antibodies is quantified. The amount ofprotein in the treated biological sample is compared with the amount inan untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.) Various types ofmicroarrays are well known and thoroughly described in DNA Microarrays:A Practical Approach, M. Schena, ed. (1999) Oxford University Press,London, hereby expressly incorporated by reference.

In another embodiment of the invention, nucleic acid sequences encodingSECP may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. Either coding or noncodingsequences may be used, and in some instances, noncoding sequences may bepreferable over coding sequences. For example, conservation of a codingsequence among members of a multi-gene family may potentially causeundesired cross hybridization during chromosomal mapping. The sequencesmay be mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries. (See, e.g., Harrington, J. J. et al.(1997) Nat. Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.) Once mapped, thenucleic acid sequences of the invention may be used to develop geneticlinkage maps, for example, which correlate the inheritance of a diseasestate with the inheritance of a particular chromosome region orrestriction fragment length polymorphism (RFLP). (See, for example,Lander, E. S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA83:7353-7357.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995)in Meyers, supra, pp. 965-968.) Examples of genetic map data can befound in various scientific journals or at the Online MendelianInheritance in Man (OMIM) World Wide Web site. Correlation between thelocation of the gene encoding SECP on a physical map and a specificdisorder, or a predisposition to a specific disorder, may help definethe region of DNA associated with that disorder and thus may furtherpositional cloning efforts.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the exact chromosomal locus is notknown. This information is valuable to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once the gene or genes responsible for a disease or syndromehave been crudely localized by genetic linkage to a particular genomicregion, e.g., ataxia-telangiectasia to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the instant invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, SECP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between SECPand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with SECP, or fragments thereof, and washed. Bound SECP is thendetected by methods well known in the art. Purified SECP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding SECP specificallycompete with a test compound for binding SECP. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with SECP.

In additional embodiments, the nucleotide sequences which encode SECPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever.

The disclosures of all patents, applications and publications, mentionedabove and below and including U.S. Ser. No. 60/247,642, U.S. Ser. No.60/249,824, U.S. Ser. No. 60/252,824, U.S. Ser. No. 60/247,505, U.S.Ser. No. 60/254,305, and U.S. Ser. No. 60/256,448, are expresslyincorporated by reference herein.

EXAMPLES I. Construction of cDNA Libraries

Incyte cDNAs were derived from cDNA libraries described in the LIFESEQGOLD database (Incyte Genomics, Palo Alto Calif.) and shown in Table 4,column 5. Some tissues were homogenized and lysed in guanidiniumisothiocyanate, while others were homogenized and lysed in phenol or ina suitable mixture of denaturants, such as TRIZOL (Life Technologies), amonophasic solution of phenol and guanidine isothiocyanate. Theresulting lysates were centrifuged over CsCl cushions or extracted withchloroform. RNA was precipitated from the lysates with eitherisopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A)+ RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Life Technologies), using the recommendedprocedures or similar methods known in the art. (See, e.g., Ausubel,1997, supra, units 5.1-6.6.) Reverse transcription was initiated usingoligo d(T) or random primers. Synthetic oligonucleotide adapters wereligated to double stranded cDNA, and the cDNA was digested with theappropriate restriction enzyme or enzymes. For most libraries, the cDNAwas size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B,or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (LifeTechnologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad Calif.), PBK-CMVplasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICISplasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto Calif.), orpINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmidswere transformed into competent E. coli cells including XL1-Blue,XL1-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10Bfrom Life Technologies.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from hostcells by in vivo excision using the UNIZAP vector system (Stratagene) orby cell lysis. Plasmids were purified using at least one of thefollowing: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96plasmid purification kit from QIAGEN. Following precipitation, plasmidswere resuspended in 0.1 ml of distilled water and stored, with orwithout lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II weresequenced as follows. Sequencing reactions were processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions were prepared using reagents provided by AmershamPharmacia Biotech or supplied in ABI sequencing kits such as the ABIPRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides were carried out using the MEGABACE1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences wereidentified using standard methods (reviewed in Ausubel, 1997, supra,unit 7.7). Some of the cDNA sequences were selected for extension usingthe techniques disclosed in Example VIII.

The polynucleotide sequences derived from Incyte cDNAs were validated byremoving vector, linker, and poly(A) sequences and by masking ambiguousbases, using algorithms and programs based on BLAST, dynamicprogramming, and dinucleotide nearest neighbor analysis. The Incyte cDNAsequences or translations thereof were then queried against a selectionof public databases such as the GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM;PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus,Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, PaloAlto Calif.); and hidden Markov model (HMM)-based protein familydatabases such as PFAM. (HMM is a probabilistic approach which analyzesconsensus primary structures of gene families. See, for example, Eddy,S. R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries wereperformed using programs based on BLAST, FASTA, BLIMPS, and HMMER. TheIncyte cDNA sequences were assembled to produce full lengthpolynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs,stitched sequences, stretched sequences, or Genscan-predicted codingsequences (see Examples IV and V) were used to extend Incyte cDNAassemblages to full length. Assembly was performed using programs basedon Phred, Phrap, and Consed, and cDNA assemblages were screened for openreading frames using programs based on GeneMark, BLAST, and FASTA. Thefull length polynucleotide sequences were translated to derive thecorresponding full length polypeptide sequences. Alternatively, apolypeptide of the invention may begin at any of the methionine residuesof the full length translated polypeptide. Full length polypeptidesequences were subsequently analyzed by querying against databases suchas the GenBank protein databases (genpept), SwissProt, the PROTEOMEdatabases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markovmodel (HMM)-based protein family databases such as PFAM. Full lengthpolynucleotide sequences are also analyzed using MACDNASIS PRO software(Hitachi Software Engineering, South San Francisco Calif.) and LASERGENEsoftware (DNASTAR). Polynucleotide and polypeptide sequence alignmentsare generated using default parameters specified by the CLUSTALalgorithm as incorporated into the MEGALIGN multisequence alignmentprogram (DNASTAR), which also calculates the percent identity betweenaligned sequences.

Table 7 summarizes the tools, programs, and algorithms used for theanalysis and assembly of Incyte cDNA and full length sequences andprovides applicable descriptions, references, and threshold parameters.The first column of Table 7 shows the tools, programs, and algorithmsused, the second column provides brief descriptions thereof, the thirdcolumn presents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

The programs described above for the assembly and analysis of fulllength polynucleotide and polypeptide sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO:64-126.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 4.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative secreted proteins were initially identified by running theGenscan gene identification program against public genomic sequencedatabases (e.g., gbpri and gbhtg). Genscan is a general-purpose geneidentification program which analyzes genomic DNA sequences from avariety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol.268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol.8:346-354). The program concatenates predicted exons to form anassembled cDNA sequence extending from a methionine to a stop codon. Theoutput of Genscan is a FASTA database of polynucleotide and polypeptidesequences. The maximum range of sequence for Genscan to analyze at oncewas set to 30 kb. To determine which of these Genscan predicted cDNAsequences encode secreted proteins, the encoded polypeptides wereanalyzed by querying against PFAM models for secreted proteins.Potential secreted proteins were also identified by homology to IncytecDNA sequences that had been annotated as secreted proteins. Theseselected Genscan-predicted sequences were then compared by BLASTanalysis to the genpept and gbpri public databases. Where necessary, theGenscan-predicted sequences were then edited by comparison to the topBLAST hit from genpept to correct errors in the sequence predicted byGenscan, such as extra or omitted exons. BLAST analysis was also used tofind any Incyte cDNA or public cDNA coverage of the Genscan-predictedsequences, thus providing evidence for transcription. When Incyte cDNAcoverage was available, this information was used to correct or confirmthe Genscan predicted sequence. Full length polynucleotide sequenceswere obtained by assembling Genscan-predicted coding sequences withIncyte cDNA sequences and/or public cDNA sequences using the assemblyprocess described in Example BT. Alternatively, full lengthpolynucleotide sequences were derived entirely from edited or uneditedGenscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data

“Stitched” Sequences

Partial cDNA sequences were extended with exons predicted by the Genscangene identification program described in Example IV. Partial cDNAsassembled as described in Example III were mapped to genomic DNA andparsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster wasanalyzed using an algorithm based on graph theory and dynamicprogramming to integrate cDNA and genomic information, generatingpossible splice variants that were subsequently confirmed, edited, orextended to create a full length sequence. Sequence intervals in whichthe entire length of the interval was present on more than one sequencein the cluster were identified, and intervals thus identified wereconsidered to be equivalent by transitivity. For example, if an intervalwas present on a cDNA and two genomic sequences, then all threeintervals were considered to be equivalent. This process allowsunrelated but consecutive genomic sequences to be brought together,bridged by cDNA sequence. Intervals thus identified were then “stitched”together by the stitching algorithm in the order that they appear alongtheir parent sequences to generate the longest possible sequence, aswell as sequence variants. Linkages between intervals which proceedalong one type of parent sequence (cDNA to cDNA or genomic sequence togenomic sequence) were given preference over linkages which changeparent type (cDNA to genomic sequence). The resultant stitched sequenceswere translated and compared by BLAST analysis to the genpept and gbpripublic databases. Incorrect exons predicted by Genscan were corrected bycomparison to the top BLAST hit from genpept. Sequences were furtherextended with additional cDNA sequences, or by inspection of genomicDNA, when necessary.

“Stretched” Sequences

Partial DNA sequences were extended to full length with an algorithmbased on BLAST analysis. First, partial cDNAs assembled as described inExample III were queried against public databases such as the GenBankprimate, rodent, mammalian, vertebrate, and eukaryote databases usingthe BLAST program. The nearest GenBank protein homolog was then comparedby BLAST analysis to either Incyte cDNA sequences or GenScan exonpredicted sequences described in Example IV. A chimeric protein wasgenerated by using the resultant high-scoring segment pairs (HSPs) tomap the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both were used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences were therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences were examined to determine whether it contained a completegene.

VI. Chromosomal Mapping of SECP Encoding Polynucleotides

The sequences which were used to assemble SEQ ID NO:64-126 were comparedwith sequences from the Incyte LIFESEQ database and public domaindatabases using BLAST and other implementations of the Smith-Watermanalgorithm. Sequences from these databases that matched SEQ ID NO:64-126were assembled into clusters of contiguous and overlapping sequencesusing assembly algorithms such as Phrap (Table 7). Radiation hybrid andgenetic mapping data available from public resources such as theStanford Human Genome Center (SHGC), Whitehead Institute for GenomeResearch (WIGR), and Généthon were used to determine if any of theclustered sequences had been previously mapped. Inclusion of a mappedsequence in a cluster resulted in the assignment of all sequences ofthat cluster, including its particular SEQ ID NO:, to that map location.

Map locations are represented by ranges, or intervals, of humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (ThecentiMorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Généthon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” World Wide Web site(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine ifpreviously identified disease genes map within or in proximity to theintervals indicated above.

VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel (1995) supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in cDNA databases such as GenBank orLIFESEQ (Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:

$\frac{{BLAST}\mspace{14mu}{Score} \times {Percent}\mspace{14mu}{Identity}}{5 \times {minimum}\mspace{14mu}\left\{ {{{length}\left( {{Seq}.\mspace{14mu} 1} \right)},{{length}\left( {{Seq}.\mspace{14mu} 2} \right)}} \right\}}$The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

Alternatively, polynucleotide sequences encoding SECP are analyzed withrespect to the tissue sources from which they were derived. For example,some full length sequences are assembled, at least in part, withoverlapping Incyte cDNA sequences (see Example III). Each cDNA sequenceis derived from a cDNA library constructed from a human tissue. Eachhuman tissue is classified into one of the following organ/tissuecategories: cardiovascular system; connective tissue; digestive system;embryonic structures; endocrine system; exocrine glands; genitalia,female; genitalia, male; germ cells; hemic and immune system; liver;musculoskeletal system; nervous system; pancreas; respiratory system;sense organs; skin; stomatognathic system; unclassified/mixed; orurinary tract. The number of libraries in each category is counted anddivided by the total number of libraries across all categories.Similarly, each human tissue is classified into one of the followingdisease/condition categories: cancer, cell line, developmental,inflammation, neurological, trauma, cardiovascular, pooled, and other,and the number of libraries in each category is counted and divided bythe total number of libraries across all categories. The resultingpercentages reflect the tissue- and disease-specific expression of cDNAencoding SECP. cDNA sequences and cDNA library/tissue information arefound in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

VIII. Extension of SECP Encoding Polynucleotides

Full length polynucleotide sequences were also produced by extension ofan appropriate fragment of the full length molecule usingoligonucleotide primers designed from this fragment. One primer wassynthesized to initiate 5′ extension of the known fragment, and theother primer was synthesized to initiate 3′ extension of the knownfragment. The initial primers were designed using OLIGO 4.06 software(National Biosciences), or another appropriate program, to be about 22to 30 nucleotides in length, to have a GC content of about 50% or more,and to anneal to the target sequence at temperatures of about 68° C. toabout 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; MolecularProbes, Eugene Oreg.) dissolved in 1×TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Corning Costar,Acton Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1 agarose gel to determine which reactions weresuccessful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Pharmacia Biotech). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase(Stratagene) to fill-in restriction site overhangs, and transfected intocompetent E. coli cells. Transformed cells were selected onantibiotic-containing media, and individual colonies were picked andcultured overnight at 37° C. in 384-well plates in LB/2× carb liquidmedia.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulfoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Applied Biosystems).

In like manner, full length polynucleotide sequences are verified usingthe above procedure or are used to obtain 5′ regulatory sequences usingthe above procedure along with oligonucleotides designed for suchextension, and an appropriate genomic library.

IX. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:64-126 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10′ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under conditions of up to, for example, 0.1× saline sodiumcitrate and 0.5% sodium dodecyl sulfate. Hybridization patterns arevisualized using autoradiography or an alternative imaging means andcompared.

X. Microarrays

The linkage or synthesis of array elements upon a microarray can beachieved utilizing photolithography, piezoelectric printing (ink-jetprinting, See, e.g., Baldeschweiler, supra.), mechanical microspottingtechnologies, and derivatives thereof. The substrate in each of theaforementioned technologies should be uniform and solid with anon-porous surface (Schena (1999), supra). Suggested substrates includesilicon, silica, glass slides, glass chips, and silicon wafers.Alternatively, a procedure analogous to a dot or slot blot may also beused to arrange and link elements to the surface of a substrate usingthermal, UV, chemical, or mechanical bonding procedures. A typical arraymay be produced using available methods and machines well known to thoseof ordinary skill in the art and may contain any appropriate number ofelements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470;Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J.Hodgson (1998) Nat. Biotechnol. 16:27-31.)

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments oroligomers thereof may comprise the elements of the microarray. Fragmentsor oligomers suitable for hybridization can be selected using softwarewell known in the art such as LASERGENE software (DNASTAR). The arrayelements are hybridized with polynucleotides in a biological sample. Thepolynucleotides in the biological sample are conjugated to a fluorescentlabel or other molecular tag for ease of detection. After hybridization,nonhybridized nucleotides from the biological sample are removed, and afluorescence scanner is used to detect hybridization at each arrayelement. Alternatively, laser desorption and mass spectrometry may beused for detection of hybridization. The degree of complementarity andthe relative abundance of each polynucleotide which hybridizes to anelement on the microarray may be assessed. In one embodiment, microarraypreparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μMdGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5(Amersham Pharmacia Biotech). The reverse transcription reaction isperformed in a 25 ml volume containing 200 ng poly(A)⁺ RNA withGEMBRIGHT kits (Incyte). Specific control poly(A)⁺ RNAs are synthesizedby in vitro transcription from non-coding yeast genomic DNA. Afterincubation at 37° C. for 2 hr, each reaction sample (one with Cy3 andanother with Cy5 labeling) is treated with 2.5 ml of 0.5M sodiumhydroxide and incubated for 20 minutes at 85° C. to the stop thereaction and degrade the RNA. Samples are purified using two successiveCHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.(CLONTECH), Palo Alto Calif.) and after combining, both reaction samplesare ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodiumacetate, and 300 ml of 100% ethanol. The sample is then dried tocompletion using a SpeedVAC (Savant Instruments Inc., Holbrook N.Y.) andresuspended in 14 μl 5×SSC/0.2% SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert. Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Pharmacia Biotech).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sites areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The sample mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope objective(Nikon, Inc., Melville N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood Mass.) installed in an IBM-compatible PC computer. The digitizeddata are displayed as an image where the signal intensity is mappedusing a linear 20-color transformation to a pseudocolor scale rangingfrom blue (low signal) to red (high signal). The data is also analyzedquantitatively. Where two different fluorophores are excited andmeasured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal within each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte).

XI. Complementary Polynucleotides

Sequences complementary to the SECP-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring SECP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of SECP. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the SECP-encoding transcript.

XII. Expression of SECP

Expression and purification of SECP is achieved using bacterial orvirus-based expression systems. For expression of SECP in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express SECP uponinduction with isopropyl. beta-D-thiogalactopyranoside (IPTG).Expression of SECP in eukaryotic cells is achieved by infecting insector mammalian cell lines with recombinant Autographica californicanuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding SECP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, SECP is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from SECP at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch. 10 and 16). Purified SECP obtained by these methods can beused directly in the assays shown in Examples XVI, XVII, and XVIII whereapplicable.

XIII. Functional Assays

SECP function is assessed by expressing the sequences encoding SECP atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, CarlsbadCalif.), both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,for example, an endothelial or hematopoietic cell line, using eitherliposome formulations or electroporation. 1-2 μg of an additionalplasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP and to evaluate the apoptotic state ofthe cells and other cellular properties. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York N.Y.

The influence of SECP on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding SECPand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding SECP and other genes of interestcan be analyzed by northern analysis or microarray techniques.

XIV. Production of SECP Specific Antibodies

SECP substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the SECP amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubel,1995, supra, ch. 11.)

Typically, oligopeptides of about 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) using FMOCchemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide and anti-SECP activity by,for example, binding the peptide or SECP to a substrate, blocking with1% BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XV. Purification of Naturally Occurring SECP Using Specific Antibodies

Naturally occurring or recombinant SECP is substantially purified byimmunoaffinity chromatography using antibodies specific for SECP. Animmunoaffinity column is constructed by covalently coupling anti-SECPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing SECP are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof SECP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/SECP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and SECPis collected.

XVI. Identification of Molecules which Interact with SECP

SECP, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton, A. E. and W. M. Hunter (1973)Biochem. J. 133:529-539.) Candidate molecules previously arrayed in thewells of a multi-well plate are incubated with the labeled SECP, washed,and any wells with labeled SECP complex are assayed. Data obtained usingdifferent concentrations of SECP are used to calculate values for thenumber, affinity, and association of SECP with the candidate molecules.

Alternatively, molecules interacting with SECP are analyzed using theyeast two-hybrid system as described in Fields, S, and O. Song (1989)Nature 340:245-246, or using commercially available kits based on thetwo-hybrid system, such as the MATCHMAKER system (Clontech).

SECP may also be used in the PATHCALLING process (CuraGen Corp., NewHaven Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins encoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

XVII. Demonstration of SECP Activity

Peroxidase activity of SECP is measured using a spectrophotometric assay(see, for example, Jeong, M. et al. (2000) J. Biol. Chem.275:2924-2930), or using an assay kit such as, for example, the AMPLEXRed Peroxidase Assay Kit from Molecular Probes together with afluorescence microplate reader or fluorometer.

An assay for growth stimulating or inhibiting activity of SECP measuresthe amount of DNA synthesis in Swiss mouse 3T3 cells (McKay, I. andLeigh, I., eds. (1993) Growth Factors: A Practical Approach, OxfordUniversity Press, New York, N.Y.). In this assay, varying amounts ofSECP are added to quiescent 3T3 cultured cells in the presence of[³H]thymidine, a radioactive DNA precursor. SECP for this assay can beobtained by recombinant means or from biochemical preparations.Incorporation of [³H]thymidine into acid-precipitable DNA is measuredover an appropriate time interval, and the amount incorporated isdirectly proportional to the amount of newly synthesized DNA. A lineardose-response curve over at least a hundred-fold SECP concentrationrange is indicative of growth modulating activity. One unit of activityper milliliter is defined as the concentration of SECP producing a 50%response level, where 100% represents maximal incorporation of[³H]thymidine into acid-precipitable DNA.

Alternatively, TGF-β activity is measured by induction of non-neoplasticnormal rat kidney fibroblasts to undergo anchorage-independent growth inthe presence of epidermal growth factor (2.5 ng/ml) as described byAssoian, R. K. et al. (1983) J. Biol. Chem. 258:7155-7160.

Alternatively, an assay for SECP activity measures the stimulation orinhibition of neurotransmission in cultured cells. Cultured CHOfibroblasts are exposed to SECP. Following endocytic uptake of SECP, thecells are washed with fresh culture medium, and a whole cellvoltage-clamped Xenopus myocyte is manipulated into contact with one ofthe fibroblasts in SECP-free medium. Membrane currents are recorded fromthe myocyte. Increased or decreased current relative to control valuesare indicative of neuromodulatory effects of SECP (Morimoto, T. et al.(1995) Neuron 15:689-696).

Alternatively, an assay for SECP activity measures the amount of SECP insecretory, membrane-bound organelles. Transfected cells as describedabove are harvested and lysed. The lysate is fractionated using methodsknown to those of skill in the art, for example, sucrose gradientultracentrifugation. Such methods allow the isolation of subcellularcomponents such as the Golgi apparatus, ER, small membrane-boundvesicles, and other secretory organelles. Immunoprecipitations fromfractionated and total cell lysates are performed using SECP-specificantibodies, and immunoprecipitated samples are analyzed using SDS-PAGEand immunoblotting techniques. The concentration of SECP in secretoryorganelles relative to SECP in total cell lysate is proportional to theamount of SECP in transit through the secretory pathway.

Alternatively, an assay for measuring protein kinase activity of SECP isperformed by quantifying the phosphorylation of a protein substrate bySECP in the presence of gamma-labeled ³²P-ATP. SECP is incubated withthe protein substrate, ³²P-ATP, and an appropriate kinase buffer. The³²P incorporated into the substrate is separated from free ³²P-ATP byelectrophoresis and the incorporated ³²P is counted using a radioisotopecounter. The amount of incorporated ³²P is proportional to the activityof SCEP. A determination of the specific amino acid residuephosphorylated is made by phosphoamino acid analysis of the hydrolyzedprotein.

Alternatively, AMP binding activity is measured by combining SECP with³²P-labeled AMP. The reaction is incubated at 37° C. and terminated byaddition of trichloroacetic acid. The acid extract is neutralized andsubjected to gel electrophoresis to remove unbound label. Theradioactivity retained in the gel is proportional to SECP activity.

XVIII. Demonstration of Immunoglobulin Activity

An assay for SECP activity measures the ability of SECP to recognize andprecipitate antigens from serum. This activity can be measured by thequantitative precipitin reaction. (Golub, E. S. et al. (1987)Immunology: A Synthesis, Sinauer Associates, Sunderland, M A, pages113-115.) SECP is isotopically labeled using methods known in the art.Various serum concentrations are added to constant amounts of labeledSECP. SECP-antigen complexes precipitate out of solution and arecollected by centrifugation. The amount of precipitable. SECP-antigencomplex is proportional to the amount of radioisotope detected in theprecipitate. The amount of precipitable SECP-antigen complex is plottedagainst the serum concentration. For various serum concentrations, acharacteristic precipitin curve is obtained, in which the amount ofprecipitable SECP-antigen complex initially increases proportionatelywith increasing serum concentration, peaks at the equivalence point, andthen decreases proportionately with further increases in serumconcentration. Thus, the amount of precipitable SECP-antigen complex isa measure of SECP activity which is characterized by sensitivity to bothlimiting and excess quantities of antigen.

Alternatively, an assay for SECP activity measures the expression ofSECP on the cell surface. cDNA encoding SECP is transfected into anon-leukocytic cell line. Cell surface proteins are labeled with biotin(de la Fuente, M. A. et. al. (1997) Blood 90:2398-2405).Immunoprecipitations are performed using SECP-specific antibodies, andimmunoprecipitated samples are analyzed using SDS-PAGE andimmunoblotting techniques. The ratio of labeled immunoprecipitant tounlabeled immunoprecipitant is proportional to the amount of SECPexpressed on the cell surface.

Alternatively, an assay for SECP activity measures the amount of cellaggregation induced by overexpression of SECP. In this assay, culturedcells such as NIH3T3 are transfected with cDNA encoding SECP containedwithin a suitable mammalian expression vector under control of a strongpromoter. Cotransfection with cDNA encoding a fluorescent markerprotein, such as Green Fluorescent Protein (CLONTECH), is useful foridentifying stable transfectants. The amount of cell agglutination, orclumping, associated with transfected cells is compared with thatassociated with untransfected cells. The amount of cell agglutination isa direct measure of SECP activity.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with certain embodiments,it should be understood that the invention as claimed should not beunduly limited to such specific embodiments. Indeed, variousmodifications of the described modes for carrying out the inventionwhich are obvious to those skilled in molecular biology or relatedfields are intended to be within the scope of the following claims.

TABLE 1 Incyte Polypeptide Incyte Polynucleo- Incyte Project SEQ IDPolypeptide tide SEQ Polynucleo- ID NO: ID ID NO: tide ID 2719959 1 2719959CD1 64  2719959CB1 7473618 2  7473618CD1 65  7473618CB1 35641363  3564136CD1 66  3564136CB1 624334 4   624334CD1 67   624334CB1 74833935  7483393CD1 68  7483393CB1 1799943 6  1799943CD1 69  1799943CB12013095 7  2013095CD1 70  2013095CB1 4674740 8  4674740CD1 71 4674740CB1 146907 9   146907CD1 72   146907CB1 1513563 10  1513563CD173  1513563CB1 3144709 11  3144709CD1 74  3144709CB1 4775686 12 4775686CD1 75  4775686CB1 5851038 13  5851038CD1 76  5851038CB171850066 14 71850066CD1 77 71850066CB1 2488934 15  2488934CD1 78 2488934CB1 2667946 16  2667946CD1 79  2667946CB1 2834555 17  2834555CD180  2834555CB1 5544174 18  5544174CD1 81  5544174CB1 1728049 19 1728049CD1 82  1728049CB1 2425121 20  2425121CD1 83  2425121CB1 281792521  2817925CD1 84  2817925CB1 4000264 22  4000264CD1 85  4000264CB14304004 23  4304004CD1 86  4304004CB1 4945912 24  4945912CD1 87 4945912CB1 7230481 25  7230481CD1 88  7230481CB1 71947526 2671947526CD1 89 71947526CB1 6843919 27  6843919CD1 90  6843919CB1 586645128  5866451CD1 91  5866451CB1 1310222 29  1310222CD1 92  1310222CB11432223 30  1432223CD1 93  1432223CB1 1537636 31  1537636CD1 94 1537636CB1 1871333 32  1871333CD1 95  1871333CB1 7153010 33  7153010CD196  7153010CB1 7996779 34  7996779CD1 97  7996779CB1 640025 35  640025CD1 98   640025CB1 1545079 36  1545079CD1 99  1545079CB1 266815037  2668150CD1 100  2668150CB1 2804787 38  2804787CD1 101  2804787CB14003882 39  4003882CD1 102  4003882CB1 4737462 40  4737462CD1 103 4737462CB1 4921634 41  4921634CD1 104  4921634CB1 6254942 42 6254942CD1 105  6254942CB1 6747838 43  6747838CD1 106  6747838CB17050585 44  7050585CD1 107  7050585CB1 3880321 45  3880321CD1 108 3880321CB1 3950005 46  3950005CD1 109  3950005CB1 3043830 47 3043830CD1 110  3043830CB1 002479 48   002479CD1 111   002479CB11395420 49  1395420CD1 112  1395420CB1 1634103 50  1634103CD1 113 1634103CB1 2422023 51  2422023CD1 114  2422023CB1 4241771 52 4241771CD1 115  4241771CB1 5046408 53  5046408CD1 116  5046408CB16271376 54  6271376CD1 117  6271376CB1 7032326 55  7032326CD1 118 7032326CB1 7078691 56  7078691CD1 119  7078691CB1 7089352 57 7089352CD1 120  7089352CB1 7284533 58  7284533CD1 121  7284533CB17482209 59  7482209CD1 122  7482209CB1 7482314 60  7482314CD1 123 7482314CB1 7482339 61  7482339CD1 124  7482339CB1 7949557 62 7949557CD1 125  7949557CB1 1555909 63  1555909CD1 126  1555909CB1

TABLE 2 GenBank Polypeptide Incyte ID NO: or SEQ ID Polypeptide PROTEOMEProbability NO: ID ID NO: Score Annotation 1  2719959CD1 g147947261.00E−176 [fl][Homo sapiens] CUB and sushi multiple domains 1 protein(Sun, P. C. et al. (2001) Genomics. 75 (1-3), 17-25) 2  7473618CD1g531385 7.80E−266 [Drosophila melanogaster] peroxidasin precursor(Nelson, R. E. et al. (1994) EMBO J. 13, 3438-3447) 3  3564136CD1g537514 1.20E−110 [Homo sapiens] arylacetamide deacetylase (Probst, M.R. et al. (1994) J. Biol. Chem. 34:21650-21656) 4  624334CD1 g5085744.70E−148 [Rattus norvegicus] neurexophilin (Petrenko, A. G. et al.(1996) J. Neurosci. 16 (14), 4360-4369) 5  7483393CD1 g132745281.00E−112 [fl][Homo sapiens] complement-c1q tumor necrosisfactor-related protein 6  1799943CD1 g164671 2.30E−36 [Sus scrofa]preprosecretin precursor (Kopin, A. S. et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87, 2299-2303) 7  2013095CD1 g3978238 2.40E−57 [Homosapiens] TNF-induced protein GG2-1 (Horrevoets, A. J. et al. (1999)Blood 93 (10), 3418-3431) 8  4674740CD1 g7271867 7.70E−26 [Homo sapiens]golgi membrane protein GP73 (Kladney, R. D. et al. (2000) Gene 249(1-2), 53-65) 26 71947526CD1 g387048 1.00E−52 [Cricetus cricetus]DHFR-coamplified protein (Foreman, P. K. et al. (1989) Mol. Cell. Biol.9, 1137-1147) 27  6843919CD1 g57736 4.50E−31 [Rattus rattus] potentialligand-binding protein (Dear, T. N. et al. (1991) EMBO J. 10 (10),2813-2819) 28  5866451CD1 g296605 7.50E−148 [Mus musculus] nodalTGF-beta like gene (Zhou, X. et al. (1993) Nature 361 (6412), 543-547)45  3880321CD1 g8572229 5.80E−22 [Homo sapiens] ubiquitous TPR-motifprotein Y isoform (Shen, P. et al. (2000) Proc. Natl. Acad. Sci. U.S.A.97 (13), 7354-7359) 46  3950005CD1 g2988399 1.50E−188 [Homo sapiens] SAgene (Loftus, B. J. et al. (1999) Genomics 60 (3), 295-308) 47 3043830CD1 g3236368 0 [Mus musculus] S3-12 (Scherer P. E. et al. (1998)Nature Biotechnol. 16:581-586) 63  1555909CD1 g4324682 3.40E−97 [Rattusnorvegicus] late gestation lung protein 1 (Kaplan, F. et al. (1999) Ant.I. Physiol. 276 (6), L1027-L1036)

TABLE 3 SEQ Incyte Amino Potential Potential Analytical ID PolypeptideAcid Phosphorylation Glycosylation Signature Sequences, Methods and NO:ID Residues Sites Sites Domains and Motifs Databases 1 2719959CD1 351S145 S151 S172 N2 N221 CUB domains: HMMER_PFAM T236 T241 T4 T59 N234N311 C54-Y159, C231-Y336 N73 Sushi domain (SCR repeat): HMMER_PFAMC170-C227 GLYCOPROTEIN DOMAIN EGFLIKE PROTEIN BLAST_PRODOM PRECURSORSIGNAL RECEPTOR INTRINSIC FACTORB12 REPEAT PD000165: C231-Y336 C1R/C1SREPEAT BLAST_DOMO DM00162|I49540|748-862: C231-Y336DM00162|I49540|592-708: C227-S338 DM00162|I49540|438-552: C231-V340DM00162|P98063|755-862: T236-Y336 2 7473618CD1 1463 S1164 S1190 N1068Signal_cleavage: SPSCAN S1315 S1320 S167 N1161 M1-P23 S171 S233 S310N1283 Signal peptide: HMMER S500 S554 S613 N1352 N271 M1-C28 S627 S634S696 N387 N401 Peroxidase domain: HMMER_PFAM S719 S871 S90 N529 N626K726-S1164 S903 S929 T1070 N705 N717 Immunoglobulin domain: HMMER_PFAMT1123 T117 T141 G248-A307, G344-A400, C440-A490, G525-A582 T225 T254 T34Leucine Rich Repeat: HMMER_PFAM T347 T389 T424 Q51-K74, N75-E98,N99-I122, S123-L146, T472 T504 T520 R147-D170, S171-L195 T53 T566 T628Leucine rich repeat C-terminal domain HMMER_PFAM T639 T710 T823 LRRCT:Y1234 Y1345 Y303 N180-Q232 von Willebrand factor type C domain:HMMER_PFAM C1395-C1450 Animal haem peroxidase signature BLIMPS_PRINTSPR00457: R751-R762, M802-T817, F954-T972, T972-W992, V997-G1023,T1050-I1060, D1177-W1197, L1248-D1262 PEROXIDASE OXIDOREDUCTASEPRECURSOR BLAST_PRODOM SIGNAL HEME GLYCOPROTEIN PROTEIN SIMILARMYELOPEROXIDASE EOSINOPHIL PD001354: K1166-F1272 PROTEIN ZK994.3 K09C8.5PEROXIDASIN BLAST_PRODOM PRECURSOR SIGNAL PD144227: N584-K726 PEROXIDASEOXIDOREDUCTASE PRECURSOR BLAST_PRODOM SIGNAL MYELOPEROXIDASE HEMEGLYCOPROTEIN ASCORBATE CATALASE LASCORBATE PD000217: Y727-A784;R825-K931; F1086-T1163 HEMICENTIN PRECURSOR SIGNAL BLAST_PRODOMGLYCOPROTEIN EGFLIKE DOMAIN HIM4 PROTEIN ALTERNATIVE SPLICING PD066634:P234-C398 MYELOPEROXIDASE DM01034|S46224|911-1352: BLAST_DOMO C859-C1298DM01034|P09933|284-735: A857-D1297 DM01034|P35419|276-725: C859-D1297DM01034|P11678|282-714: F862-Q1296 VWFC domain signature: MOTIFSC1414-C1450 3 3564136CD1 401 S100 S119 S231 N282 N323 ARYLACETAMIDEDEACETYLASE EC 3.1.1. BLAST_PRODOM S30 S395 T102 AADAC HYDROLASETRANSMEMBRANE T255 T80 T85 MICROSOME SIGNAL ANCHOR Y297 PD087155:E207-D314 PD087138: G2-R105 PROTEIN HYDROLASE PUTATIVE ESTERASEBLAST_PRODOM C4A8.06C CHROMOSOME I N-ACETYL PHOSPHINO THRICIN TRIPETIDEDEACETYLASE COSMID B1740 PD150195: T102-L194 Lipolytic enzymes “G-D-X-G”family, BLIMPS_BLOCKS histidine BL01173: V107-S119, V140-F166, R182-A195signal peptide signal_peptide: HMMER M1-T21 Spscan signal_cleavage:SPSCAN M1-F19 4 624334CD1 271 S37 S49 S83 T112 N146 N156 NEUREXOPHILINNEUROPHILIN BLAST_PRODOM T130 T138 T182 N162 N23 PD039440: S83-G271 T41T62 T70 Y261 N68 N93 PD123274: M1-Y82 Spscan signal_cleavage: SPSCANM1-G27 5 7483393CD1 201 S178 S65 T98 signal_peptide: HMMER M1-P18signal_cleavage: SPSCAN M1-G15 Complement protein C1q domain HMMER_PFAMC1q: A63-V190 C1q domain proteins. BLIMPS_BLOCKS BL01113: G30-C56,P80-A115, A147-Q166, S183-S192 Complement C1Q domain signatureBLIMPS_PRINTS PR00007: F101-A120, A147-G168, T181-Y191, P74-K100 C1QDOMAIN BLAST_DOMO DM00777|Q02105|71-245: P29-D193DM00777|P98085|222-418: G30-D193 DM00777|P23206|477-673: P29-V190DM00777|S23297|465-674: P29-L189 C1QB PRECURSOR SIGNAL COLLAGEN REPEATBLAST_PRODOM HYDROXYLATION GLYCOPROTEIN CHAIN PLASMA EXTRACELLULARMATRIX PD002992: A63-V190 6 1799943CD1 121 S29 S58 T117 signal_peptide:HMMER M1-A18 signal_cleavage: SPSCAN M1-A18 Peptide hormone HMMER_PFAMhormone2: H28-G55 Glucagon/GIP/secretin/VIP family BLIMPS_BLOCKSBL00260: H28-V54 GLUCAGON POLYPEPTIDE HORMONE BLIMPS_PRINTS PR00275:H28-S38, R39-L50 BRAIN NATRIURETIC PEPTIDE BLIMPS_PRINTS PR00712C:L46-N64 Glucagon/GIP/secretin/VIP family MOTIFS signature: H28-L50 72013095CD1 186 S5 S52 T136 T34 signal_cleavage: SPSCAN M1-S36 84674740CD1 436 S277 S328 S36 N115 N150 signal_peptide: HMMER S366 S68S92 M1-A29 T195 T312 T76 signal_cleavage: SPSCAN Y399 M1-A29transmembrane_domain: HMMER G11-N31 9 146907CD1 134 T49 S50 S55signal_peptide: HMMER M101-L129 10 1513563CD1 172 T142 S3 S50signal_peptide: HMMER M7-G36 11 3144709CD1 80 signal_peptide: HMMERM1-S19 12 4775686CD1 92 T29 T36 signal_peptide: HMMER M1-S21 135851038CD1 90 S37 signal_peptide: HMMER M1-G21 14 71850066CD1 354 S12S133 S15 N129 N163 signal_cleavage: SPSCAN S192 S195 S52 M1-S15 S71 T213T314 KTI12 PROTEIN ATPBINDING BLAST_PRODOM PD040436: M1-P110ATP/GTP-binding site motif A (P-loop): MOTIFS G8-S15 15 2488934CD1 101S20 signal_peptide: HMMER M1-S21 signal_cleavage: SPSCAN M1-M22 162667946CD1 74 S11 T40 N14 signal_peptide: HMMER M1-A31 signal_cleavage:SPSCAN M1-T40 Sodium: solute symporter family signature PROFILESCANsodium_symporters_1.prf: P9-F52 17 2834555CD1 100 S47 T50signal_peptide: HMMER M1-G21 18 5544174CD1 94 S2 S59 signal_peptide:HMMER M1-S22 signal_cleavage: SPSCAN M1-A65 19 1728049CD1 143 S128 S90T83 N81 signal_peptide: HMMER M1-A27 signal_cleavage: SPSCAN M1-G35 202425121CD1 116 S2 S48 S97 signal_peptide: HMMER M1-A25 signal_cleavage:SPSCAN M1-R28 21 2817925CD1 76 S15 T18 T37 signal_peptide: HMMER M1-R20signal_cleavage: SPSCAN M1-C39 22 4000264CD1 116 S61 T111signal_peptide: HMMER M1-G27 signal_cleavage: SPSCAN M1-G29 234304004CD1 210 S116 S120 S39 signal_cleavage: SPSCAN S88 T123 T131M1-G41 T15 T205 Y132 transmembrane_domain: HMMER Y18-W38 24 4945912CD1195 S128 T131 T181 signal_cleavage: SPSCAN M1-A58 25 7230481CD1 140 S103S3 signal_peptide: HMMER M1-A19 Actinin-type actin-binding domainPROFILESCAN signatures actinin_2.prf: N48-Q94 26 71947526CD1 585 S136S263 Y73 N106 N189 signal_cleavage: SPSCAN S265 S281 T91 N220 N315M1-R37 S352 S532 S63 N89 transmembrane_domain: HMMER S550 S78 T104K13-A33 T317 T35 T359 Aminotransferases class-V pyridoxal- MOTIFS T371T376 phosphate attachment site: L312-I329 27 6843919CD1 95 S68 T22 T41signal_peptide: HMMER M1-G23 signal_cleavage: SPSCAN M1-G23 UTEROGLOBINFAMILY BLAST_DOMO DM02636|S17449|1-94: M1-D93 POTENTIAL LIGAND BINDINGPROTEIN RYD5 BLAST_PRODOM PD065166: M1-D93 UTEROGLOBIN SIGNATUREBLIMPS_PRINTS PR00486A: K2-C16 28 5866451CD1 347 S127 S219 S83 N199 N72Signal_cleavage: SPSCAN S99 M1-G33 Signal_peptide: HMMER M1-A25 TGF-betafamily signature MOTIFS I265-C280 Transforming growth factor beta likeHMMER_PFAM TGF-beta: C247-L347 TGF-beta family signature tgf_beta.prf:PROFILESCAN Q245-K301 TGF-beta family proteins BLIMPS_BLOCKS BL00250:C247-N282, T311-C346 GROWTH FACTOR CYSTINE KN BLIMPS_PRINTS PR00438:N272-P281, E342-C346 GLYCOPROTEIN PRECURSOR SIGNAL GROWTH BLAST_PRODOMFACTOR PD000357: C247-C346 NODAL PRECURSOR DEVELOPMENTAL BLAST_PRODOMPROTEIN GROWTH FACTOR PD117903: M1-P53 TGF-BETA FAMILY BLAST_DOMODM00245|P43021|34-354: G33-L347 DM00245|P48970|64-383: S244-C346,F77-W162 DM00245|I49541|105-420: K233-C346, P51-R157DM00245|P12644|95-408: K233-C346, P51-R157 29 1310222CD1 63Signal_cleavage: SPSCAN M1-R19 30 1432223CD1 208 Signal_cleavage: SPSCANM1-N65 PROTEIN COX4AL F25H2.4 PD022799: A8-I195 BLAST_PRODOM 311537636CD1 256 S131 S236 S30 Signal_cleavage: SPSCAN S69 S9 T172 T194M1-G54 T215 32 1871333CD1 229 S172 S225 T23 N148 Signal_cleavage: SPSCANT26 T85 M1-G19 Signal_peptide: HMMER M1-A20 Transmembrane domain: HMMERL3-G22, F56A8.1 PROTEIN BLAST_PRODOM PD146797: E33-K214 33 7153010CD1327 S126 S213 S307 N172 N311 Signal_cleavage: SPSCAN T23 M1-S19Signal_peptide: HMMER M1-V21 Immunoglobulin domain ig: HMMER_PFAMG57-V144, C187-A239 CELL PRECURSOR GLYCOPROTEIN BLAST_PRODOMTRANSMEMBRANE SIGNAL IMMUNOGLOBULIN FOLD ADHESION ALTERNATIVE SPLICINGPD005007: W44-G201 MYELIN; SCHWANN; SIALOADHESIN; FORM; BLAST_DOMODM03744|P20138|1-142: W44-T165 34 7996779CD1 104 S45 Signal_cleavage:SPSCAN M1-G30 Signal_peptide: HMMER M1-G30 35 640025CD1 82 S51Signal_cleavage: SPSCAN M1-A35 Signal_peptide: HNMER M34-S51 361545079CD1 367 S117 S21 T327 N285 Signal_cleavage: SPSCAN Y219 M1-A63Leucine zipper pattern MOTIFS L346-L367 SUA5/yciO/yrdC family prBLIMPS_BLOCKS BL01147: V170-V194, L228-M241, L251-P263 Signal_peptide:HMMER M89-S117 HMM_score 17.56 SUA5/yciO/yrdC family Sua5_yciO_yrd:HMMER_PFAM V162-G343 PROTEIN HYPF TRANSCRIPTIONAL BLAST_PRODOMREGULATORY DNABINDING ZINCFINGER CONSERVED INTERGENIC PD002209:A163-S332 HYPOTHETICAL SUA5/YCIO/YRDC FAMILY BLAST_DOMODM02523|P45831|25-166: A163-E296 DM02523|P45103|1-206: L158-G343DM02523|P39153|26-169: A163-E296 DM02523|P45847|1-217: L158-S332 372668150CD1 70 S50 S52 T45 N59 Signal_cleavage: SPSCAN M1-R25Signal_peptide: HMMER M1-R25 Transmembrane domain: HMMER I6-V23, 382804787CD1 73 N67 Signal_peptide: HMMER M1-G23 Signal_cleavage: SPSCANM1-S65 Transmembrane domain: HMMER L4-I21, 39 4003882CD1 76 S64 T67Signal_cleavage: SPSCAN M1-S65 Leucine zipper pattern MOTIFS L26-L47,L30-L51 40 4737462CD1 80 S36 S50 Signal_cleavage: SPSCAN M1-G21Signal_peptide: HMMER M1-G22 41 4921634CD1 73 S63 Signal_cleavage:SPSCAN M1-S17 Signal_peptide: HMMER M1-C22 Transmembrane domain: HMMERM1-F25, 42 6254942CD1 116 S11 S3 T17 Signal_cleavage: SPSCAN M1-A42Transmembrane domain: HMMER I49-A66 43 6747838CD1 95 S54 S64 S80Signal_peptide: HMMER M1-A18 44 7050585CD1 138 S131 T121 T64Signal_cleavage: SPSCAN T73 M1-L49 Signal_peptide: HMMER M1-W18 453880321CD1 134 S46 S59 S65 Signal_cleavage: SPSCAN M1-S32 46 3950005CD1570 S195 S254 S339 N269 N288 Putative AMP-binding domain signatureMOTIFS S479 S504 S525 N476 N82 I227-K238 S64 S91 S99 T150Signal_peptide: HMMER T262 T345 T362 M1-C20 T544 T84 Y464 AMP-bindingenzyme AMP-binding: HMMER_PFAM S91-V502 Putative AMP-binding domainsignature PROFILESCAN amp_binding.prf: E209-V259 AMP-BINDING SIGNATUREBLIMPS_PRINTS PR00154: R222-T233, T234-H242 LIGASE SYNTHETASE PROTEINENZYME BLAST_PRODOM BIOSYNTHESIS MULTIFUNCTIONAL REPEAT ACYLCOAPD000070: T147-V421 SA PROTEIN GENE SIGNAL KIDNEY SPECIFIC BLAST_PRODOMPD151238: V49-W90 PUTATIVE AMP-BINDING DOMAIN BLAST_DOMODM00073|A61209|65-538: E67-Q402, G417-K561 DM00073|P39062|50-555:K89-K561 DM00073|P27550|82-615: F203-K561, L66-D170DM00073|P27095|107-644: R197-K561, G70-V276 47 3043830CD1 1325Signal_cleavage: SPSCAN M1-A32 SUBMAXILLARY APOMUCIN ICE NUCLEATIONBLAST_PRODOM PROTEIN FILAMENTOUS HEMAGGLUTININ ANTIGEN S312 PD011940:T82-T996 PROTEIN PERILIPIN ADIPOSE BLAST_PRODOM DIFFERENTIATION RELATEDADRP MEMBRANE CARGO SELECTION TIP47 A/B PD018256: P1135-F1318 S312BLAST_PRODOM PD185810: M1-L112 PROTEIN F36H2.3A F36H2.3B BLAST_PRODOMPD004794: L251-T1048 SURFACE; S-LAYER; ARRAY; SAPA2; BLAST_DOMODM08156|A56143|1-932: G28-V877 ICE NUCLEATION PROTEIN BLAST_DOMODM00787|P18127|603-942: G507-G855 DM00787|P06620|194-533: V481-Q802 48002479CD1 228 S44 S165 S187 signal_cleavage: SPSCAN S207 T62 T83 M1-R46T214 49 1395420CD1 80 S74 N10 signal_cleavage: SPSCAN M1-S58 GHMPkinases putative ATP-binding PROFILESCAN domain: R3-N69 50 1634103CD1538 S220 S489 S522 signal_cleavage: SPSCAN T105 T464 M1-A35transmembrane domain: HMMER P127-T150 NICOTINATE PHOSPHO BLAST_PRODOMRIBOSYLTRANSFERASE TRANSFERASE GLYCOSYLTRANSFERASE PD008895: E268-L434,F92-E223 PD011757: L16-L80 51 2422023CD1 73 T25 signal_cleavage: SPSCANM1-G19 signal peptide: HMMER M1-G19 52 4241771CD1 108 S89 S102 N33signal_cleavage: SPSCAN M1-C24 signal peptide: HMMER M1-P26 535046408CD1 80 N15 signal_cleavage: SPSCAN M1-G19 signal peptide: HMMERM1-G19 54 6271376CD1 87 S18 S38 S43 S47 signal_cleavage: SPSCAN M1-A15signal peptide: HMMER M1-S18 55 7032326CD1 78 S5 S76 signal_cleavage:SPSCAN M1-A27 signal peptide: HMMER M1-G29 56 7078691CD1 108 S60 S75signal_cleavage: SPSCAN M1-C19 signal peptide: HMMER M1-G21 577089352CD1 81 S27 S42 S49 S78 signal_cleavage: SPSCAN M1-A26 signalpeptide: HMMER M1-A26 58 7284533CD1 146 S107 T101 T122 signal_cleavage:SPSCAN T123 M1-A62 signal peptide: HMMER M1-G27 59 7482209CD1 92 S17 S59T21 T81 N71 signal_cleavage: SPSCAN M1-A16 signal peptide: HMMER M1-S1960 7482314CD1 119 S100 T90 T113 signal peptide: HMMER M50-R81 617482339CD1 92 S58 N41 signal_cleavage: SPSCAN M1-S24 signal peptide:HMMER M1-S24 62 7949557CD1 107 S34 S89 S105 signal_cleavage: SPSCANM1-T27 transmembrane domain: HMMER I5-L22 63 1555909CD1 497 S75 S130S201 N27 N41 signal_cleavage: SPSCAN S228 S279 S362 N451 M1-G22 S453S471 T29 signal peptide: HMMER T81 T170 T179 M1-G22 T184 T241 T467SCP-like extracellular protein: HMMER_PFAM T483 Y392 K56-G208Extracellular proteins SCP/Tpx-1/Ag5/PR- BLIMPS_BLOCKS 1/Sc7 proteinsBL01009: M80-C97, H127-Y140, T160-C180, V194-E209 Allergen V5/Tpx-1family signature BLIMPS_PRINTS PR00837: H127-Y140, C159-C175, Y195-G208,M80-I98 Venom allergen 5 signature BLIMPS_PRINTS PR00838: A50-L66,M80-I98, G125-Y140, M158-V177 PROTEIN PRECURSOR SIGNAL BLAST_PRODOMPATHOGENESISRELATED ANTIGEN ALLERGEN VENOM MULTIGENE FAMILY AG5PD000542: R67-G208, R53-G227 FSG 120K CYSRICH PROTEIN GLYCOPROTEINBLAST_PRODOM EGF LIKE DOMAIN PD128352: I51-G226 EXTRACELLULAR PROTEINSBLAST_DOMO SCP/TPX-1/AG5/PR-1/SC7 DM00332|P48060|1-175: N41-W206DM00332|P35778|12-207: D55-P211 DM00332|Q03401|9-181: K56-G208DM00332|Q05110|34-223: V47-Y212 Extracellular proteins SCP/Tpx-1/Ag5/PR-MOTIFS 1/Sc7 signature 2 Y195-W206

TABLE 4 Polynucleotide Incyte Sequence Selected 5′ 3′ SEQ ID NO:Polynucleotide ID Length Fragment(s) Sequence Fragments PositionPosition 64 2719959CB1 1338 1-363, 1269-1338 56002879J1 1 984 2719959T6(LUNGTUT10) 724 1338 65 7473618CB1 5093 1-1579, 4240-4299, 6866460F8(BRAGNON02) 315 550 2099-3946, 72341159D1 4076 4718 4379-4529GBI.g8152129_000001.edit 3942 4373 GBI.g8152129_000003.edit 2447 3944g1547765 3947 4380 7754154H1 (HEAONOE01) 331 1094FL7473618_g8096904_000020_g7292259 2031 3945 GBI.g8152037_000006.edit2 1550 7754154J1 (HEAONOE01) 946 1622 72342123D1 4260 5093 55081807J1 36804019 GBI.g8096904_10_14_4_9_20.2.edit 912 2177 66 3564136CB1 1392 1-242,478-673 GBI.g8954235.order_0.edit 1 1041 2352447H1 (COLSUCT01) 784 988g1525737 937 1392 3564136H1 (SKINNOT05) 144 451 g1493356 224 495g1678558 674 1260 67 624334CB1 2390 710-1069, 2366-2390, 71392568V1 302792 1-245 4338525F6 (BRAUNOT02) 1 453 71199569V1 1831 2380 g1210731 17872390 6273383F8 (BRAIFEN03) 584 1331 7130272H1 (BRAHTDK01) 1423 19186447629H1 (BRAINOC01) 1186 1822 68 7483393CB1 3248 1-2012 71275974V1 1638 71870255V1 1722 2386 5895459F8 (BRAYDIN03) 2588 3248 72032402V1 6081438 8225765H1 (COLHTUS02) 2658 3248 71870671V1 1535 2084 72335020V12354 3247 71066648V1 1000 1634 69 1799943CB1 520 1-87, 231-520GBI.g6715656_000011.edit.3 1 213 1799943T6 (COLNNOT27) 137 520 702013095CB1 2108 134-424, 1-71 7724892J1 (THYRDIE01) 1 685 8126837H1(SCOMDIC01) 562 1050 70284485V1 1275 1954 70285683V1 1504 2108 2456045F6(ENDANOT01) 870 1304 71 4674740CB1 2219 1855-2219 55048995J1 381 1261(ADMEDNV37) 7468169H1 (LUNGNOE02) 1 496 7979128H1 (LSUBDMC01) 1448 221955048913J1 620 1564 (ADMEDNV37) 72 146907CB1 1678 270-1678, 1-7371157131V1 519 1192 144826R1 (TLYMNOR01) 664 1259 71156479V1 1108 167871156776V1 1 651 73 1513563CB1 2374 1-1026 72106415V1 1268 208272106477V1 1208 1963 72106501V1 1607 2374 7463376H1 (LIVRFEE04) 1 55772105630V1 570 1234 72105342V1 530 1198 74 3144709CB1 842 38-60, 804-8426728561H1 (COLITUT02) 1 670 2837521H2 (DRGLNOT01) 606 842 75 4775686CB1837 175-300, 806-837 7156574H1 (ESOGTUR02) 86 772 805170H1 (BSTMNOT01) 1208 4775686F6 (BRAQNOT01) 431 837 76 5851038CB1 828 398-762 55022063J1442 828 (GPCRDNV87) g2629754 1 397 5851038F7 (FIBAUNT02) 142 6615851038H1 (FIBAUNT02) 141 386 77 71850066CB1 1696 1-653 71638522V1 3961014 5996956H1 (BRAZDIT04) 1103 1696 71635790V1 851 1407 2518629F6(BRAITUT21) 1 478 71636467V1 473 1047 78 2488934CB1 841 1-218 2488934T6(KIDNTUT13) 225 841 2488934F6 (KIDNTUT13) 1 537 79 2667946CB1 27521-566, 2730-2752, 71668418V1 895 1663 749-909 8244690H1 (BONEUNR01) 1666 71669177V1 1764 2417 71667244V1 2159 2752 71664085V1 1447 222571664868V1 646 1282 80 2834555CB1 934 512-934, 1-55, 7002906H1(COLNFEC01) 399 934 201-272 3189343R6 (THYMNON04) 1 556 81 5544174CB1815 176-481, 61-82 5544174F6 (TESTNOC01) 289 815 6953446F8 (BRAITDR02) 1641 82 1728049CB1 1242 513-962, 1-185 724829R6 (SYNOOAT01) 1 6736822418J1 (SINTNOR01) 502 1230 1728049F6 (PROSNOT14) 799 1239 4803643H1(MYEPUNT01) 997 1242 83 2425121CB1 4217 1-1656, 4170-4217 1511561F6(LUNGNOT14) 1969 2533 55146378J1 1 863 1293328F1 (PGANNOT03) 3908 41762291068R6 (BRAINON01) 3123 3718 842419R6 (PROSTUT05) 2707 3199 3108255F6(BRSTTUT15) 903 1559 7171832H1 (BRSTTMC01) 1473 2025 1621469T6(BRAITUT13) 3593 4169 6812454H1 (ADRETUR01) 2113 2680 1739860R6(HIPONON01) 3416 3888 3931569H1 (PROSTUT09) 3982 4217 6997857R8(BRAXTDR17) 573 1209 7582572H1 (BRAIFEC01) 1722 2108 70681972V1 26162968 84 2817925CB1 1301 1-490, 893-1231 7414958T1 (PITUNON01) 178 8441888610F6 (BLADTUT07) 855 1301 6305824T6 (NERDTDN03) 1 827 8242705J1(BONEUNR01) 630 1188 85 4000264CB1 2148 1790-2148, 550-1393 7458107H1(LIVRTUE01) 1575 2148 6753255H1 (SINTFER02) 280 780 71384040V1 1 3807071128H1 (BRAUTDR02) 563 1162 7022226H1 (PANCNON03) 1000 1640 7724208H1(THYRDIE01) 1443 2045 86 4304004CB1 1141 961-1141, 376-493, 4304004F8(BRSTTUT18) 1 553 1-28 70465082V1 497 1141 87 4945912CB1 855 80-355,831-855 4945912F8 (SINTNOT25) 1 522 71146178V1 638 852 8031651J1(TESTNOF01) 397 851 g1941671 485 855 88 7230481CB1 617 1-362 7230481F8(BRAXTDR15) 1 617 89 71947526CB1 2460 1218-1314 71265535V1 1884 246071947895V1 736 1561 3776352F6 (BRSTNOT27) 1604 2291 71682330V1 1503 224371947074V1 1 828 72431962D1 816 1588 90 6843919CB1 431 6843919H1(KIDNTMN03) 1 431 91 5866451CB1 1050 1-191 GNN.g7264172_00003_002 1 10447317786R8 (BRAWTDK01) 707 1050 92 1310222CB1 1822 1-221 1417610F1(KIDNNOT09) 487 1141 SANA03735F1 1173 1822 2383314F6 (ISLTNOT01) 1 562604946H1 (BRSTTUT01) 1553 1822 1467420F1 (PANCTUT02) 606 1242 931432223CB1 855 1432223H1 (BEPINON01) 1 222 1476162T6 (LUNGTUT03) 188 8491630467F6 (COLNNOT19) 373 855 94 1537636CB1 1440 1416-1440 801691H1(BRAVTXT04) 1 264 7059329H1 (BRALNON02) 9 730 g1191911 985 14403181951T6 (TLYJNOT01) 799 1326 194915T6 (KIDNNOT02) 416 1098 951871333CB1 1389 1-20, 1360-1389, 71129962V1 871 1389 756-855 71142771V1600 1210 71132064V1 543 1135 71179205V1 1 608 96 7153010CB1 1500 1-134,920-971, 6934671F6 (SINTTMR02) 537 1273 1373-1500, 419-753, 6934671R6(SINTTMR02) 775 1500 1239-1276 7152316F6 (BONEUNR01) 1 668 97 7996779CB1796 1-63, 185-796 5687774H1 (BRAIUNT01) 1 198 7996779H1 (ADRETUC01) 53796 98 640025CB1 2540 1-50 8077582J1 (ADRETUE02) 1 765 7639394H1(SEMVTDE01) 1366 2059 8324134J1 (MIXDUNN04) 2253 2529 7440482H1(ADRETUE02) 502 1123 g1186398 1836 2540 70673692V1 2264 2540 5506313R6(BRADDIR01) 922 1412 7637348H1 (SINTDIE01) 1473 2075 5422789T6(PROSTMT07) 1975 2524 99 1545079CB1 2487 1-315 6302525H1 (UTREDIT07) 266596 1545079T6 (PROSTUT04) 1802 2471 7345625H1 (SYNODIN02) 649 11794103346F6 (BRSTTUT17) 450 1019 2457841F6 (ENDANOT01) 1754 2303 066132H1(HUVESTB01) 1 264 1970803H1 (UCMCL5T01) 206 487 5599584H1 (UTRENON03)2055 2487 1364772R6 (SCORNON02) 1249 1810 6456268H1 (COLNDIC01) 11381748 100 2668150CB1 701 1-110 7341082T8 (COLNDIN02) 1 701 101 2804787CB11956 1-39, 507-614, 70749428V1 791 1441 1014-1454 g2166802 1 60170749393V1 194 829 70745592V1 963 1504 70054082D1 1388 1956 1024003882CB1 1063 1-1063 70788074V1 521 1063 70792833V1 1 618 1034737462CB1 495 1-98, 146-495 4737462F6 (THYMNOR02) 1 495 104 4921634CB1880 674-880, 450-482 4921634F6 (TESTNOT11) 1 588 70803614V1 322 880 1056254942CB1 2666 2610-2666, 1-580 1943214T6 (HIPONOT01) 1956 26497744938H1 (ADRETUE04) 1025 1626 6476322H1 (PROSTMC01) 2237 26668133916H1 (SCOMDIC01) 626 1276 7991669H2 (UTRSDIC01) 1 510 6345860H1(LUNGDIS03) 387 712 1258806F6 (MENITUT03) 2219 2657 1271246F1(TESTTUT02) 1459 2140 106 6747838CB1 1293 1-145, 654-1293 g4266852 258653 6747838F8 (BRAXNOT03) 675 1293 6891936H1 (BRAITDR03) 1 522GBI.g7960452.edit 1 1293 107 7050585CB1 693 1-693 7050539H1 (BRACNOK02)1 693 7050539R8 (BRACNOK02) 1 693 108 3880321CB1 860 1-509, 787-86071880126V1 1 600 71883910V1 280 860 109 3950005CB1 2738 722-1030,2409-2738 70770220V1 1321 1894 4082341F6 (CONFNOT02) 2266 2738 4081043F8(CONFNOT02) 1167 1624 70775991V1 442 1049 6837615H1 (BRSTNON02) 20482422 5276224H1 (MUSLNOT01) 1662 1910 4795834F8 (LIVRTUT09) 1060 160271346657V1 1 592 3175849T6 (UTRSTUT04) 1820 2369 70776014V1 672 1166 1103043830CB1 6108 1-3559 6902402H1 (MUSLTDR02) 5094 5582 7174759H1(BRSTTMC01) 3289 3958 7174777H1 (BRSTTMC01) 2657 3342 2775475F6(PANCNOT15) 1599 2218 8225152H1 (COLHTUS02) 4437 5131 1964133R6(BRSTNOT04) 4379 5124 55024920H1 1 693 (PKINDNV13) 7689084J1 (PROSTME06)5474 6108 55026065J1 596 1309 (PKINDNV23) 7173660H2 (BRSTTMC01) 24663033 2690419F6 (LUNGNOT23) 3855 4423 3541678H1 (SEMVNOT04) 3678 40151961558H1 (BRSTNOT04) 4066 4425 55025178J1 1033 1846 (PKINDNV15)3690484F6 (HEAANOT01) 1908 2583 111 002479CB1 1110 1-836 70111790V1 5601110 70111692V1 1 613 112 1395420CB1 1902 1521-1902, 1-27 70501084V11003 1462 8175577H1 (FETANOA01) 298 828 7234467H1 (BRAXTDR15) 859 14293033671F6 (TLYMNOT05) 1299 1902 7730353R6 (UTRCDIE01) 340 997 5891913H1(UTRENOT06) 1 318 113 1634103CB1 1960 305-324, 1-265 6824111H1(SINTNOR01) 1 499 7339828H1 (SINTNON02) 1408 1960 6753665J1 (SINTFER02)326 1069 71264720V1 1078 1752 1815281F6 (PROSNOT20) 1182 1774 1634103F6(COLNNOT19) 586 1156 114 2422023CB1 540 517-540 2422023T6 (SCORNON02) 1508 2244504R6 (HIPONON02) 168 540 115 4241771CB1 1321 1-1023, 1301-132172582414V1 500 1321 6013180F8 (FIBRUNT02) 1 629 116 5046408CB1 536 1-5365046408F8 (PLACFER01) 1 535 5046408H1 (PLACFER01) 249 536 117 6271376CB11345 1-38, 1238-1345, 4864015F8 (PROSTUT09) 1 660 933-983 8083757H1(BRACDIK08) 621 1345 118 7032326CB1 1060 403-1060 6800476R8 (COLENOR03)371 1060 6800476F8 (COLENOR03) 1 653 119 7078691CB1 1192 113-11926262640F8 (MCLDTXN03) 491 1192 7078691H1 (BRAUTDR04) 1 579 1207089352CB1 693 1-554 7089352F7 (BRAUTDR03) 1 693 121 7284533CB1 8881-340, 761-888 7284533H1 (BRAIFEJ01) 342 888 7284533R8 (BRAIFEJ01) 2 5827284533F8 (BRAIFEJ01) 1 508 122 7482209CB1 618 480-618 7470241H1(LUNGNOE02) 97 618 g6989749 1 479 123 7482314CB1 755 1-78, 198-225,g2055889 226 755 667-755 6435849F8 (LUNGNON07) 1 420 124 7482339CB1 386g1833238 1 386 125 7949557CB1 524 1-79, 191-524 7949557J1 (BRABNOE02) 1524 126 1555909CB1 3836 1-2343, 3746-3836 1004107R1 (BRSTNOT03) 34033741 5000814F8 (PROSTUT21) 148 690 7687354H1 (PROSTME06) 968 15951506470F6 (BRAITUT07) 2630 3218 3236711F6 (COLNUCT03) 1904 24347042338H1 (UTRSTMR02) 1437 1953 5138056H1 (OVARDIT04) 3543 37915191912H1 (OVARDIT06) 3170 3432 1555909T1 (BLADTUT04) 2255 27887166118H1 (PLACNOR01) 1658 2199 7632327H1 (BLADTUE01) 629 1297 3979568H1(LUNGTUT08) 3493 3753 7403782H1 (SINIDME01) 361 817 g1645738 3515 38363675191H1 (PLACNOT07) 1 288 4947920H1 (SINTNOT25) 2222 2475 1686339H1(PROSNOT15) 3239 3462

TABLE 5 Polynucleotide SEQ ID NO: Incyte Project ID: RepresentativeLibrary 64  2719959CB1 LUNGTUT10 65  7473618CB1 HEAONOE01 66  3564136CB1SKINNOT05 67   624334CB1 BRAXNOT02 68  7483393CB1 BRADDIR01 69 1799943CB1 COLNNOT27 70  2013095CB1 TESTNOT03 71  4674740CB1 ADMEDNV3772   146907CB1 TLYMNOR01 73  1513563CB1 BRAINOT11 74  3144709CB1DRGLNOT01 75  4775686CB1 BRAQNOT01 76  5851038CB1 FIBAUNT02 7771850066CB1 URETTUE01 78  2488934CB1 KIDNTUT13 79  2667946CB1 UTRENOT0980  2834555CB1 THYMNON04 81  5544174CB1 BRAITDR02 82  1728049CB1PROSNOT14 83  2425121CB1 BLADNOT06 84  2817925CB1 BRSTNOT14 85 4000264CB1 HNT2AZS07 86  4304004CB1 PROSTUT08 87  4945912CB1 SINTNOT2588  7230481CB1 BRAXTDR15 89 71947526CB1 SINTNOT22 90  6843919CB1KIDNTMN03 91  5866451CB1 BRAWTDK01 92  1310222CB1 COLNFET02 93 1432223CB1 COLNNOT19 94  1537636CB1 BRABDIR01 95  1871333CB1 LIVRTUT1296  7153010CB1 BONEUNR01 97  7996779CB1 ADRETUC01 98   640025CB1BRSTNOT03 99  1545079CB1 ENDANOT01 100  2668150CB1 COLNDIN02 101 2804787CB1 BLADTUT08 102  4003882CB1 LUNLTUE01 103  4737462CB1THYMNOR02 104  4921634CB1 TESTNOT11 105  6254942CB1 KIDNNOT05 106 6747838CB1 BRAXNOT03 107  7050585CB1 BRACNOK02 108  3880321CB1OVARNON03 109  3950005CB1 CONFNOT02 110  3043830CB1 BRSTNOT07 111  002479CB1 U937NOT01 112  1395420CB1 THYRNOT03 113  1634103CB1STOMFET01 114  2422023CB1 SCORNON02 115  4241771CB1 LATRTUT02 116 5046408CB1 PLACFER01 117  6271376CB1 PROSTUT09 118  7032326CB1COLENOR03 119  7078691CB1 MCLDTXN03 120  7089352CB1 BRAUTDR03 121 7284533CB1 BRAIFEJ01 122  7482209CB1 LUNGNOE02 123  7482314CB1LUNGNON07 125  7949557CB1 BRABNOE02 126  1555909CB1 PLACFER01

TABLE 6 Library Vector Library Description ADMEDNV37 pCR2-TopoTA Librarywas constructed using pooled cDNA from 111 different donors. cDNA wasgenerated using mRNA isolated from pooled skeletal muscle tissue removedfrom 10 Caucasian male and female donors, ages 21-57, who died fromsudden death; from pooled thymus tissue removed from 9 Caucasian maleand female donors, ages 18-32, who died from sudden death; from pooledfetal liver tissue removed from 32 Caucasian male and female fetuses,ages 18-24 weeks, who died from spontaneous abortions; from pooled fetalkidney tissue removed from 59 Caucasian male and female fetuses, ages20-33 weeks, who died from spontaneous abortions; and from fetal braintissue removed from a 23-week-old Caucasian male fetus who died fromfetal demise. ADRETUC01 PSPORT1 This large size fractionated library wasconstructed using pooled cDNA from two donors. cDNA was generated usingmRNA isolated from adrenal gland tissue removed from an 8-year-old Blackmale (donor A), who died from anoxia and from adrenal tumor tissueremoved from a 52-year-old Caucasian female (donor B) during aunilateral adrenalectomy. For donor A, serologies were negative. Patientmedications included DDAVP, Versed, and labetalol. For donor B,pathology indicated a pheochromocytoma. Patient history included benignhypertension, depressive disorder, chronic sinusitis, idiopathicproctocolitis, a cataract, and urinary tract infection. Previoussurgeries included a vaginal hysterectomy. Patient medications includedProcardia (one dose only) and Prozac for 5 years. Family historyincluded secondary Parkinsonism in the father; cerebrovascular disease,secondary Parkinsonism and anxiety state in the mother; and benignhypertension, atherosclerotic coronary artery disease, hyperlipidemia,and brain cancer in the sibling(s). BLADNOT06 pINCY Library wasconstructed using RNA isolated from the posterior wall bladder tissueremoved from a 66-year-old Caucasian male during a radicalprostatectomy, radical cystectomy and urinary diversion. Pathology forthe associated tumor tissue indicated grade 3 transitional cellcarcinoma on the anterior wall of the bladder and urothelium. Patienthistory included lung neoplasm, and tobacco abuse in remission. Familyhistory included a malignant breast neoplasm, tuberculosis,cerebrovascular disease, atherosclerotic coronary artery disease, andlung cancer. BLADTUT08 pINCY Library was constructed using RNA isolatedfrom bladder tumor tissue removed from a 72-year-old Caucasian maleduring a radical cystectomy and prostatectomy. Pathology indicated aninvasive grade 3 (of 3) transitional cell carcinoma in the right bladderbase. Patient history included pure hypercholesterolemia and tobaccoabuse. Family history included myocardial infarction, cerebrovasculardisease, and brain cancer. BONEUNR01 PCDNA2.1 This random primed librarywas constructed using pooled cDNA from two different donors. cDNA wasgenerated using mRNA isolated from an untreated MG-63 cell line derivedfrom an osteosarcoma tumor removed from a 14-year-old Caucasian male(donor A) and using mRNA isolated from sacral bone tumor tissue removedfrom an 18-year-old Caucasian female (donor B) during an exploratorylaparotomy and soft tissue excision. Pathology indicated giant celltumor of the sacrum in donor B. Donor B's history included pelvic jointpain, constipation, urinary incontinence, unspecified abdominal/pelvicsymptoms, and a pelvic soft tissue malignant neoplasm. Family historyincluded prostate cancer in donor B. BRABDIR01 pINCY Library wasconstructed using RNA isolated from diseased cerebellum tissue removedfrom the brain of a 57-year-old Caucasian male, who died from acerebrovascular accident. Patient history included Huntington's disease,emphysema, and tobacco abuse. BRABNOE02 PBK-CMV This 5′ biased randomprimed library was constructed using RNA isolated from vermis tissueremoved from a 35-year-old Caucasian male who died from cardiac failure.Pathology indicated moderate leptomeningeal fibrosis and multiplemicroinfarctions of the cerebral neocortex. Patient history includeddilated cardiomyopathy, congestive heart failure, cardiomegaly, and anenlarged spleen and liver. Patient medications included simethicone,Lasix, Digoxin, Colace, Zantac, captopril, and Vasotec. BRACNOK02PSPORT1 This amplified and normalized library was constructed using RNAisolated from posterior cingulate tissue removed from an 85-year-oldCaucasian female who died from myocardial infarction and retroperitonealhemorrhage. Pathology indicated atherosclerosis, moderate to severe,involving the circle of Willis, middle cerebral, basilar and vertebralarteries; infarction, remote, left dentate nucleus; and amyloid plaquedeposition consistent with age. There was mild to moderateleptomeningeal fibrosis, especially over the convexity of the frontallobe. There was mild generalized atrophy involving all lobes. The whitematter was mildly thinned. Cortical thickness in the temporal lobes,both maximal and minimal, was slightly reduced. The substantia nigrapars compacta appeared mildly depigmented. Patient history includedCOPD, hypertension, and recurrent deep venous thrombosis. 6.4 millionindependent clones from this amplified library were normalized in oneround using conditions adapted Soares et al., PNAS (1994) 91:9228-9232and Bonaldo et al., Genome Research 6 (1996):791. BRADDIR01 pINCYLibrary was constructed using RNA isolated from diseased choroid plexustissue of the lateral ventricle, removed from the brain of a 57-year-oldCaucasian male, who died from a cerebrovascular accident. BRAIFEJ01PRARE This random primed 5′ cap isolated library was constructed usingRNA isolated from brain tissue removed from a Caucasian male fetus whodied at 23 weeks' gestation from premature birth. Serologies werenegative. Family history included diabetes in the mother. BRAINOT11pINCY Library was constructed using RNA isolated from brain tissueremoved from the right temporal lobe of a 5-year-old Caucasian maleduring a hemispherectomy. Pathology indicated extensive polymicrogyriaand mild to moderate gliosis (predominantly subpial and subcortical),consistent with chronic seizure disorder. Family history included acervical neoplasm. BRAITDR02 PCDNA2.1 This random primed library wasconstructed using RNA isolated from allocortex, neocortex, anterior andfrontal cingulate tissue removed from a 55-year-old Caucasian female whodied from cholangiocarcinoma. Pathology indicated mild meningealfibrosis predominately over the convexities, scattered axonal spheroidsin the white matter of the cingulate cortex and the thalamus, and a fewscattered neurofibrillary tangles in the entorhinal cortex and theperiaqueductal gray region. Pathology for the associated tumor tissueindicated well-differentiated cholangiocarcinoma of the liver withresidual or relapsed tumor. Patient history included cholangiocarcinoma,post-operative Budd-Chiari syndrome, biliary ascites, hydrothorax,dehydration, malnutrition, oliguria and acute renal failure. Previoussurgeries included cholecystectomy and resection of 85% of the liver.BRAQNOT01 pINCY Library was constructed using RNA isolated from midbraintissue removed from a 35- year-old Caucasian male. No neuropathology wasfound. Patient history included dilated cardiomyopathy, congestive heartfailure, and an enlarged spleen and liver. BRAUTDR03 PCDNA2.1 Thisrandom primed library was constructed using RNA isolated from pooledglobus pallidus and substantia innominata tissue removed from a55-year-old Caucasian female who died from cholangiocarcinoma. Pathologyindicated mild meningeal fibrosis predominately over the convexities,scattered axonal spheroids in the white matter of the cingulate cortexand the thalamus, and a few scattered neurofibrillary tangles in theentorhinal cortex and the periaqueductal gray region. Pathology for theassociated tumor tissue indicated well-differentiated cholangiocarcinomaof the liver with residual or relapsed tumor. Patient history includedcholangiocarcinoma, post-operative Budd-Chiari syndrome, biliaryascites, hydrothorax, dehydration, malnutrition, oliguria and acuterenal failure. Previous surgeries included cholecystectomy and resectionof 85% of the liver. BRAWTDK01 PSPORT1 This amplified and normalizedlibrary was constructed using RNA isolated from dentate nucleus tissueremoved from a 55-year-old Caucasian female who died fromcholangiocarcinoma. Pathology indicated no diagnostic abnormalities inthe brain or intracranial vessels. There was mild meningeal fibrosispredominately over the convexities There were scattered axonal spheroidsin the white matter of the cingulate cortex and thalamus. There were afew scattered neurofibrillary tangles in the entorhinal cortex andperiaqueductal gray region. Pathology for the associated tumor tissueindicated well-differentiated cholangiocarcinoma of the liver withresidual or relapsed tumor, surrounded by foci of bile lakes beneath thehepatic surface scar. The liver had extensive surface scarring,congestion, cholestasis, hemorrhage, necrosis, and chronic inflammation.The patient presented with nausea, vomiting, dehydration, malnutrition,oliguria, and acute renal failure. Patient history includedpost-operative Budd-Chiari syndrome, biliary ascites, bilateral acutebronchopneumonia with microabscesses, hydrothorax, and bilateral legpitting edema. Previous surgeries included cholecystectomy, liverresection, hysterectomy, bilateral salpingo-oophorectomy, and portocavalshunt. The patient was treated with a nasogastic feeding tube, biliarydrainage stent, paracentesis, pleurodesis and abdominal ultrasound.Patient medications included Ampicillin, niacin, furosemide, Aldactone,Benadryl, and morphine. Independent clones from this amplified librarywere normalized in one round using conditions adapted from Soares etal., PNAS (1994) 91:9228-9232 and Bonaldo et al., Genome Research 6(1996):791. BRAXNOT02 pINCY Library was constructed using RNA isolatedfrom cerebellar tissue removed from a 64-year-old male. Patient historyincluded carcinoma of the left bronchus. BRAXNOT03 pINCY Library wasconstructed using RNA isolated from sensory-motor cortex tissue obtainedfrom the brain of a 35- year-old Caucasian male who died from cardiacfailure. Pathology indicated moderate leptomeningeal fibrosis andmultiple microinfarctions of the cerebral neocortex. Patient historyincluded dilated cardiomyopathy, congestive heart failure, cardiomegalyand an enlarged spleen and liver. BRAXTDR15 PCDNA2.1 This random primedlibrary was constructed using RNA isolated from superior parietalneocortex tissue removed from a 55-year-old Caucasian female who diedfrom cholangiocarcinoma. Pathology indicated mild meningeal fibrosispredominately over the convexities, scattered axonal spheroids in thewhite matter of the cingulate cortex and the thalamus, and a fewscattered neurofibrillary tangles in the entorhinal cortex and theperiaqueductal gray region. Pathology for the associated tumor tissueindicated well-differentiated cholangiocarcinoma of the liver withresidual or relapsed tumor. Patient history included cholangiocarcinoma,post-operative Budd-Chiari syndrome, biliary ascites, hydrothorax,dehydration, malnutrition, oliguria and acute renal failure. Previoussurgeries included cholecystectomy and resection of 85% of the liver.BRSTNOT03 PSPORT1 Library was constructed using RNA isolated fromdiseased breast tissue removed from a 54-year-old Caucasian femaleduring a bilateral radical mastectomy. Pathology for the associatedtumor tissue indicated residual invasive grade 3 mammary ductaladenocarcinoma. Patient history included kidney infection and condylomaacuminatum. Family history included benign hypertension, hyperlipidemiaand a malignant neoplasm of the colon. BRSTNOT07 pINCY Library wasconstructed using RNA isolated from diseased breast tissue removed froma 43-year-old Caucasian female during a unilateral extended simplemastectomy. Pathology indicated mildly proliferative fibrocystic changeswith epithelial hyperplasia, papillomatosis, and duct ectasia. Pathologyfor the associated tumor tissue indicated invasive grade 4, nucleargrade 3 mammary adenocarcinoma with extensive comedo necrosis. Familyhistory included epilepsy, cardiovascular disease, and type II diabetes.BRSTNOT14 pINCY Library was constructed using RNA isolated from breasttissue removed from a 62- year-old Caucasian female during a unilateralextended simple mastectomy. Pathology for the associated tumor tissueindicated an invasive grade 3 (of 4), nuclear grade 3 (of 3)adenocarcinoma, ductal type. Ductal carcinoma in situ, comedo type,comprised 60% of the tumor mass. Metastatic adenocarcinoma wasidentified in one (of 14) axillary lymph nodes with no perinodalextension. The tumor cells were strongly positive for estrogen receptorsand weakly positive for progesterone receptors. Patient history includeda benign colon neoplasm, hyperlipidemia, cardiac dysrhythmia, andobesity. Family history included atherosclerotic coronary arterydisease, myocardial infarction, colon cancer, ovarian cancer, lungcancer, and cerebrovascular disease. COLENOR03 PCDNA2.1 Library wasconstructed using RNA isolated from colon epithelium tissue removed froma 13-year-old Caucasian female who died from a motor vehicle accident.COLNDIN02 pINCY This normalized library was constructed from 4.72million independent clones from a diseased colon and colon polyp tissuelibrary. Starting RNA was made from pooled cDNA from two donors. cDNAwas generated using mRNA isolated from diseased colon tissue removedfrom the cecum and descending colon of a 16-year-old Caucasian male(donor A) during partial colectomy, temporary ileostomy, and colonoscopyand from diseased colon polyp tissue removed from the cecum of a67-year-old female (donor B). Pathology indicated innumerable (greaterthan 100) adenomatous polyps with low-grade dysplasia involving theentire colonic mucosa in the setting of familial polyposis coli (donorA), and a benign cecum polyp (donor B). Pathology for the associatedtumor tissue (B) indicated invasive grade 3 adenocarcinoma that arose intubulovillous adenoma forming a fungating mass in the cecum. The tumorinfiltrated just through the muscularis propria. Multiple (2 of 17)regional lymph nodes were involved by metastatic adenocarcinoma. Atubulovillous adenoma and multiple (6) tubular adenomas with low-gradedysplasia were observed in the cecum and ascending colon. Donor Apresented with abdominal pain and flatulence. The patient was not takingany medications. Family history included benign colon neoplasm in thefather and sibling(s); benign hypertension, cerebrovascular disease,breast cancer, uterine cancer, and type II diabetes in thegrandparent(s). COLNFET02 pINCY Library was constructed using RNAisolated from the colon tissue of a Caucasian female fetus, who died at20 week' gestation. COLNNOT19 pINCY Library was constructed using RNAisolated from the cecal tissue of an 18-year-old Caucasian female. Thececal tissue, along with the appendix and ileum tissue, were removedduring bowel anastomosis. Pathology indicated Crohn's disease of theileum, involving 15 cm of the small bowel. COLNNOT27 pINCY Library wasconstructed using RNA isolated from diseased cecal tissue removed from31-year-old Caucasian male during a total intra-abdominal colectomy,appendectomy, and permanent ileostomy. Pathology indicated severe activeCrohn's disease involving the colon from the cecum to the rectum. Therewere deep rake-like ulcerations which spared the intervening mucosa. Theulcers extended into the muscularis, and there was transmuralinflammation. Patient history included an irritable colon. Previoussurgeries included a colonscopy. CONFNOT02 pINCY Library was constructedusing RNA isolated from abdominal fat tissue removed from a 52-year-oldCaucasian female during an ileum resection and incarcerated ventralhernia repair. Patient history included diverticulitis. Family historyincluded hyperlipidemia. DRGLNOT01 pINCY Library was constructed usingRNA isolated from dorsal root ganglion tissue removed from the cervicalspine of a 32-year-old Caucasian male who died from acute pulmonaryedema and bronchopneumonia, bilateral pleural and pericardial effusions,and malignant lymphoma (natural killer cell type). Patient historyincluded probable cytomegalovirus, infection, hepatic congestion andsteatosis, splenomegaly, hemorrhagic cystitis, thyroid hemorrhage, andBell's palsy. Surgeries included colonoscopy, large intestine biopsy,adenotonsillectomy, and nasopharyngeal endoscopy and biopsy; treatmentincluded radiation therapy. ENDANOT01 PBLUESCRIPT Library wasconstructed using RNA isolated from aortic endothelial cell tissue froman explanted heart removed from a male during a heart transplant.FIBAUNT02 pINCY Library was constructed using RNA isolated fromuntreated aortic adventitial fibroblasts obtained from a 65- year-oldCaucasian female. HEAONOE01 PCDNA2.1 This 5′ biased random primedlibrary was constructed using RNA isolated from the aorta of a39-year-old Caucasian male, who died from a gunshot wound. Serology waspositive for cytomegalovirus (CMV). Patient history included tobaccoabuse (one pack of cigarettes per day for 25 years), and occasionallycocaine, marijuana, and alcohol use. HNT2AZS07 PSPORT1 This subtractedlibrary was constructed from RNA isolated from an hNT2 cell line(derived from a human teratocarcinoma that exhibited propertiescharacteristic of a committed neuronal precursor) treated for three dayswith 0.35 micromolar AZ. The hybridization probe for subtraction wasderived from a similarly constructed library from untreated hNT2 cells.3.08M clones from the AZ-treated library were subjected to three roundsof subtractive hybridization with 3.04M clones from the untreatedlibrary. Subtractive hybridization conditions were based on themethodologies of Swaroop et al. (NAR (1991) 19:1954) and Bonaldo et al.(Genome Research (1996) 6:791). KIDNNOT05 PSPORT1 Library wasconstructed using RNA isolated from the kidney tissue of a 2-day-oldHispanic female, who died from cerebral anoxia. Family history includedcongenital heart disease. KIDNTMN03 pINCY This normalized kidney tissuelibrary was constructed from 2.08 million independent clones from a poolof two libraries from two different donors. Starting RNA was made fromright kidney tissue removed from an 8-year-old Caucasian female (donorA) who died from a motor vehicle accident and left kidney medulla andcortex tissue removed from a 53-year-old Caucasian female (donor B)during a nephroureterectomy. In donor B, pathology for the matched tumortissue indicated grade 2 renal cell carcinoma involving the lower poleof the kidney. Medical history included hyperlipidemia, cardiacdysrhythmia, metrorrhagia, normal delivery, cerebrovascular disease, andatherosclerotic coronary artery disease in donor B. The library wasnormalized in two rounds using conditions adapted from Soares et al.,PNAS (1994) 91:9228-9232 and Bonaldo et al., Genome Research 6(1996):791, except that a significantly longer (48 hours/round)reannealing hybridization was used. KIDNTUT13 pINCY Library wasconstructed using RNA isolated from kidney tumor tissue removed from a51-year-old Caucasian female during a nephroureterectomy. Pathologyindicated a grade 3 renal cell carcinoma. Patient history includeddepressive disorder, hypoglycemia, and uterine endometriosis. Familyhistory included calculus of the kidney, colon cancer, and type IIdiabetes. LATRTUT02 pINCY Library was constructed using RNA isolatedfrom a myxoma removed from the left atrium of a 43-year-old Caucasianmale during annuloplasty. Pathology indicated atrial myxoma. Patienthistory included pulmonary insufficiency, acute myocardial infarction,atherosclerotic coronary artery disease, hyperlipidemia, and tobaccouse. Family history included benign hypertension, acute myocardialinfdrction, atherosclerotic coronary artery disease, and type IIdiabetes. LIVRTUT12 pINCY Library was constructed using RNA isolatedfrom a treated C3A hepatocyte cell line, which is a derivative of HepG2, a cell line derived from a hepatoblastoma removed from a 15-year-oldCaucasian male. The cells were treated with 3- methylcholanthrene (MCA),5 mM for 48 hours. LUNGNOE02 PSPORT This 5′ biased random primed librarywas constructed using RNA isolated from lung tissue removed from a35-year-old Caucasian female during who died from a cerebrovascularaccident. Serologies were negative. Patient history includedmononucleosis, high blood pressure during pregnancies and alcohol use.LUNGNON07 pINCY This normalized lung tissue library was constructed from5.1 million independent clones from a lung tissue library. Starting RNAwas made from RNA isolated from lung tissue. The library was normalizedin two rounds using conditions adapted from Soares et al., PNAS (1994)91:9228-9232 and Bonaldo et al., Genome Research (1996) 6:791, exceptthat a significantly longer (48 hours/round) reannealing hybridizationwas used. LUNGTUT10 pINCY Library was constructed using RNA isolatedfrom lung tumor tissue removed from the left upper lobe of a 65-year-oldCaucasian female during a segmental lung resection. Pathology indicateda metastatic grade 2 myxoid liposarcoma and a metastatic grade 4liposarcoma. Patient history included soft tissue cancer, breast cancer,and secondary lung cancer. LUNLTUE01 PCDNA2.1 This 5′ biased randomprimed library was constructed using RNA isolated from left upper lobelung tumor tissue removed from a 56-year-old Caucasian male duringcomplete pneumonectomy, pericardectomy and regional lymph node excision.Pathology indicated grade 3 squamous cell carcinoma forming a mass inthe left upper lobe centrally. The tumor extended through pleura intoadjacent pericardium. Patient history included hemoptysis and tobaccoabuse. Family history included benign hypertension, cerebrovascularaccident, atherosclerotic coronary artery disease in the mother;prostate cancer in the father; and type II diabetes in the sibling(s).MCLDTXN03 pINCY This normalized dendritic cell library was constructedfrom one million independent clones from a pool of two derived dendriticcell libraries. Starting libraries were constructed using RNA isolatedfrom untreated and treated derived dendritic cells from umbilical cordblood CD34+ precursor cells removed from a male. The cells were derivedwith granulocyte/macrophage colony stimulating factor (GM-CSF), tumornecrosis factor alpha (TNF alpha), and stem cell factor (SCF). TheGM-CSF was added at time 0 at 100 ng/ml, the TNF alpha was added at time0 at 2.5 ng/ml, and the SCF was added at time 0 at 25 ng/ml. Incubationtime was 13 days. The treated cells were then exposed to phorbolmyristate acetate (PMA), and ionomycin. The PMA and ionomycin were addedat 13 days for five hours. The library was normalized in two roundsusing conditions adapted from Soares et al., PNAS (1994) 91:9228-9232and Bonaldo et al., Genome Research (1996) 6:791, except that asignificantly longer (48 hours/round) reannealing hybridization wasused. OVARNON03 pINCY This normalized ovarian tissue library wasconstructed from 5 million independent clones from an ovary library.Starting RNA was made from ovarian tissue removed from a 36-year-oldCaucasian female during total abdominal hysterectomy, bilateralsalpingo-oophorectomy, soft tissue excision, and an incidentalappendectomy. Pathology for the associated tumor tissue indicated oneintramural and one subserosal leiomyomata of the myometrium. Theendometrium was proliferative phase. Patient history included deficiencyanemia, calculus of the kidney, and a kidney anomaly. Family historyincluded hyperlipidemia, acute myocardial infarction, atheroscleroticcoronary artery disease, type II diabetes, and chronic liver disease.The library was normalized in two rounds using conditions adapted fromSoares et al., PNAS (1994) 91:9228 and Bonaldo et al., Genome Research(1996) 6:791, except that a significantly longer (48 hours/round)reannealing hybridization was used. PLACFER01 pINCY The library wasconstructed using RNA isolated from placental tissue removed from aCaucasian fetus, who died after 16 weeks' gestation from fetal demiseand hydrocephalus. Patient history included umbilical cord wrappedaround the head (3 times) and the shoulders (1 time). Serology waspositive for anti-CMV. Family history included multiple pregnancies andlive births, and an abortion. PLACFER01 pINCY The library wasconstructed using RNA isolated from placental tissue removed from aCaucasian fetus, who died after 16 weeks' gestation from fetal demiseand hydrocephalus. Patient history included umbilical cord wrappedaround the head (3 times) and the shoulders (1 time). Serology waspositive for anti-CMV. Family history included multiple pregnancies andlive births, and an abortion. PROSNOT14 pINCY Library was constructedusing RNA isolated from diseased prostate tissue removed from a60-year-old Caucasian male during radical prostatectomy and regionallymph node excision. Pathology indicated adenofibromatous hyperplasia.Pathology for the associated tumor tissue indicated an adenocarcinoma(Gleason grade 3 + 4). The patient presented with elevated prostatespecific antigen (PSA). Patient history included a kidney cyst andhematuria. Family history included benign hypertension, cerebrovasculardisease, and arteriosclerotic coronary artery disease. PROSTUT08 pINCYLibrary was constructed using RNA isolated from prostate tumor tissueremoved from a 60-year-old Caucasian male during radical prostatectomyand regional lymph node excision. Pathology indicated an adenocarcinoma(Gleason grade 3 + 4). Adenofibromatous hyperplasia was also present.The patient presented with elevated prostate specific antigen (PSA).Patient history included a kidney cyst, and hematuria. Family historyincluded tuberculosis, cerebrovascular disease, and arterioscleroticcoronary artery disease. PROSTUT09 pINCY Library was constructed usingRNA isolated from prostate tumor tissue removed from a 66-year-oldCaucasian male during a radical prostatectomy, radical cystectomy, andurinary diversion. Pathology indicated grade 3 transitional cellcarcinoma. The patient presented with prostatic inflammatory disease.Patient history included lung neoplasm, and benign hypertension. Familyhistory included a malignant breast neoplasm, tuberculosis,cerebrovascular disease, atherosclerotic coronary artery disease andlung cancer. SCORNON02 PSPORT1 This normalized spinal cord library wasconstructed from 3.24M independent clones from the a spinal cord tissuelibrary. RNA was isolated from the spinal cord tissue removed from a71-year-old Caucasian male who died from respiratory arrest. Patienthistory included myocardial infarction, gangrene, and end stage renaldisease. The normalization and hybridization conditions were adaptedfrom Soares et al. (PNAS (1994) 91:9228). SINTNOT22 pINCY Library wasconstructed using RNA isolated from small intestine tissue removed froma 15-year-old Caucasian female who died from a closed head injury.Serology was positive for cytomegalovirus. Patient history includedseasonal allergies. SINTNOT25 pINCY The library was constructed usingRNA isolated from smallintestine tissue removed from a 13-year-oldCaucasian male, who died from a gunshotwound to the head. Family historyincluded diabetes. SKINNOT05 pINCY Library was constructed using RNAisolated from skin tissue removed from a Caucasian male fetus, who diedfrom Patau's syndrome (trisomy 13) at 20-weeks' gestation. STOMFET01pINCY Library was constructed using RNA isolated from the stomach tissueof a Caucasian female fetus, who died at 20 weeks' gestation. TESTNOT03PBLUESCRIPT Library was constructed using RNA isolated from testiculartissue removed from a 37-year-old Caucasian male, who died from liverdisease. Patient history included cirrhosis, jaundice, and liverfailure. TESTNOT11 pINCY Library was constructed using RNA isolated fromtesticular tissue removed from a 16-year-old Caucasian male who diedfrom hanging. Patient history included drug use (tobacco, marijuana, andcocaine use), and medications included Lithium, Ritalin, and Paxil.THYMNON04 PSPORT1 This normalized library was constructed from a thymustissue library. Starting RNA was made from thymus tissue removed from a3-year-old Caucasian male, who died from anoxia. Serologies werenegative. The patient was not taking any medications. The library wasnormalized in two rounds using conditions adapted from Soares et al.,PNAS (1994) 91:9228 and Bonaldo et al., Genome Research (1996) 6:791,except that a significantly longer (48-hours/round) reannealinghybridization was used. THYMNOR02 pINCY The library was constructedusing RNA isolated from thymus tissue removed from a 2-year-oldCaucasian female during a thymectomy and patch closure of leftatrioventricular fistula. Pathology indicated there was no grossabnormality of the thymus. The patient presented with congenital heartabnormalities. Patient history included double inlet left ventricle anda rudimentary right ventricle, pulmonary hypertension, cyanosis,subaortic stenosis, seizures, and a fracture of the skull base. Familyhistory included reflux neuropathy. THYRNOT03 pINCY Library wasconstructed using RNA isolated from thyroid tissue removed from the leftthyroid of a 28-year-old Caucasian female during a completethyroidectomy. Pathology indicated a small nodule of adenomatoushyperplasia present in the left thyroid. Pathology for the associatedtumor tissue indicated dominant follicular adenoma, forming awell-encapsulated mass in the left thyroid. TLYMNOR01 PBLUESCRIPTLibrary was constructed using RNA isolated from non-adherent peripheralblood mononuclear cells obtained from a 24-year-old Caucasian male. Thecells were purified on Ficoll Hypaque, then harvested, lysed in GuSCN,and spun through CsCl to obtain RNA for library construction. U937NOT01PBLUESCRIPT Library was constructed at Stratagene (STR937207), using RNAisolated from the U937 monocyte-like cell line. This line (ATCC CRL1593)was established from malignant cells obtained from the pleural effusionof a 37-year-old Caucasian male with diffuse histiocytic lymphoma.URETTUE01 PCDNA2.1 This 5′ biased random primed library was constructedusing RNA isolated from ureter tumor tissue removed from a 64-year-oldCaucasian male during closed bladder biopsy, radical cystectomy, radicalprostatectomy, and formation of a cutanious ureterostomy. Pathologyindicated in situ and superficially invasive transitional cell carcinomapresenting as 2 separate papillary lesions, one located 7.5 cm from theureter margin, and the other in the right proximal ureter extending intothe renal pelvis. The tumor invaded just into the submucosal tissue. Theureter margin was involved by focal in situ transitional cell carcinoma.The patient presented with carcinoma in situ of the bladder, malignantneoplasm of the ureter, and secondary malignant kidney neoplasm. Patienthistory included malignant bladder neoplasm, psoriasis, chronic airwayobstruction, testicular hypofunction, and tobacco abuse. Previoussurgeries included appendectomy and transurethral destruction of bladderlesion. Patient medications included naproxen, Atrovent, albuterol, andan unspecified psoriasis cream. Family history included malignantstomach neoplasm in the father and malignant bladder neoplasm in thesibling(s). UTRENOT09 pINCY Library was constructed using RNA isolatedfrom endometrial tissue removed from a 38-year-old Caucasian femaleduring total abdominal hysterectomy, exploratory laparotomy, cystocelerepair, and incidental appendectomy. Patient history included missedabortion, hypertrophy of breast, bronchitis, and an unspecified closedfracture. Previous surgeries included dilation and curettage. Familyhistory included polymyositis and muliple myeloma.

TABLE 7 Program Description Reference Parameter Threshold ABI FACTURA Aprogram that removes vector sequences and Applied Biosystems, FosterCity, CA. masks ambiguous bases in nucleic acid sequences. ABI/PARACELFDF A Fast Data Finder useful in comparing and Applied Biosystems,Foster City, CA; Mismatch <50% annotating amino acid or nucleic acidsequences. Paracel Inc., Pasadena, CA. ABI AutoAssembler A program thatassembles nucleic acid sequences. Applied Biosystems, Foster City, CA.BLAST A Basic Local Alignment Search Tool useful in Altschul, S. F. etal. (1990) J. Mol. Biol. ESTs: Probability value = 1.0E−8 sequencesimilarity search for amino acid and 215:403-410; Altschul, S. F. et al.(1997) or less; Full Length sequences: nucleic acid sequences. BLASTincludes five Nucleic Acids Res. 25:3389-3402. Probability value =1.0E−10 or functions: blastp, blastn, blastx, tblastn, and tblastx. lessFASTA A Pearson and Lipman algorithm that searches for Pearson, W. R.and D. J. Lipman (1988) ESTs: fasta E value = 1.06E−6; similaritybetween a query sequence and a group of Proc. Natl. Acad Sci. USA85:2444- Assembled ESTs: fasta Identity = sequences of the same type.FASTA comprises as 2448; Pearson, W. R. (1990) Methods 95% or greaterand Match least five functions: fasta, tfasta, fastx, tfastx, andEnzymol. 183:63-98; and Smith, T. F. length = 200 bases or greater;ssearch. and M. S. Waterman (1981) Adv. Appl. fastx E value = 1.0E−8 orless; Math. 2:482-489. Full Length sequences: fastx score = 100 orgreater BLIMPS A BLocks IMProved Searcher that matches a Henikoff, S.and J. G. Henikoff (1991) Probability value = 1.0E−3 or sequence againstthose in BLOCKS, PRINTS, Nucleic Acids Res. 19:6565-6572; less DOMO,PRODOM, and PFAM databases to Henikoff, J. G. and S. Henikoff (1996)search for gene families, sequence homology, and Methods Enzymol.266:88-105; and structural fingerprint regions. Attwood, T. K. et al.(1997) J. Chem. Inf. Comput. Sci. 37:417-424. HMMER An algorithm forsearching a query sequence Krogh, A. et al. (1994) J. Mol. Biol. PFAMhits: Probability value = against hidden Markov model (HMM)-based235:1501-1531; Sonnhammer, E. L. L. 1.0E−3 or less; Signal peptidedatabases of protein family consensus sequences, et al. (1988) NucleicAcids Res. 26: hits: Score = 0 or greater such as PFAM. 320-322; Durbin,R. et al. (1998) Our World View, in a Nutshell, Cambridge Univ. Press,pp. 1-350. ProfileScan An algorithm that searches for structural andGribskov, M. et al. (1988) CABIOS Normalized quality score ≧ GCG-sequence motifs in protein sequences that match 4:61-66; Gribskov, M. etal. (1989) specified “HIGH” value for that sequence patterns defined inProsite. Methods Enzymol.183:146-159; Bairoch, particular Prosite motif.A. et al. (1997) Nucleic Acids Res. Generally, score = 1.4-2.1.25:217-221. Phred A base-calling algorithm that examines automatedEwing, B. et al. (1998) Genome Res. sequencer traces with highsensitivity and 8:175-185; Ewing, B. and P. Green probability. (1998)Genome Res. 8:186-194. Phrap A Phils Revised Assembly Program includingSmith, T. F. and M. S. Waterman (1981) Score = 120 or greater; MatchSWAT and CrossMatch, programs based on Adv. Appl. Math. 2:482-489;Smith, length = 56 or greater efficient implementation of theSmith-Waterman T. F. and M. S. Waterman (1981) J. Mol. algorithm, usefulin searching sequence Biol. 147:195-197; and Green, P., homology andassembling DNA sequences. University of Washington, Seattle, WA. ConsedA graphical tool for viewing and editing Phrap Gordon, D. et al. (1998)Genome Res. assemblies. 8:195-202. SPScan A weight matrix analysisprogram that scans Nielson, H. et al. (1997) Protein Score = 3.5 orgreater protein sequences for the presence of secretory Engineering10:1-6; Claverie, J. M. and signal peptides. S. Audic (1997) CABIOS12:431-439. TMAP A program that uses weight matrices to delineatePersson, B. and P. Argos (1994) J. Mol. transmembrane segments onprotein sequences Biol. 237:182-192; Persson, B. and P. and determineorientation. Argos (1996) Protein Sci. 5:363-371. TMHMMER A program thatuses a hidden Markov model Sonnhammer, E. L. et al. (1998) Proc. (HMM)to delineate transmembrane segments on Sixth Intl. Conf. On IntelligentSystems protein sequences and determine orientation. for Mol. Biol.,Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence (AAAI)Press, Menlo Park, CA, and MIT Press, Cambridge, MA, pp. 175-182. MotifsA program that searches amino acid sequences for Bairoch, A. et al.(1997) Nucleic Acids patterns that matched those defined in Prosite.Res. 25:217-221; Wisconsin Package Program Manual, version 9, pageM51-59, Genetics Computer Group, Madison, WI.

What is claimed is:
 1. An isolated humanized or chimeric antibody whichspecifically binds to a polypeptide consisting of amino acids H28-G55 ofSEQ ID No:
 6. 2. The antibody of claim 1, wherein the antibody is achimeric antibody.
 3. The antibody of claim 1 conjugated to a carrierprotein.
 4. The antibody of claim 3, wherein the carrier protein isselected from the group consisting of bovine serum albumin,thyroglobulin, and keyhole limpet hemocyanin.
 5. A compositioncomprising the antibody of claim 1 and at least one pharmaceuticallyacceptable excipient.
 6. The composition of claim 5, wherein theantibody is labeled.
 7. The composition of claim 6, wherein the label isa reporter molecule or an enzyme that is capable of generating ameasurable signal.
 8. The composition of claim 6, wherein the label isselected from the group consisting of radioactive isotopes, ligands,chemiluminescent agents, and enzymes.
 9. The antibody of claim 1,wherein the antibody is a humanized antibody.
 10. The antibody of claim1, wherein the antibody is a single chain antibody.
 11. The antibody ofclaim 1, wherein the antibody is a Fab fragment.
 12. The antibody ofclaim 1, wherein the antibody is a F(ab′)₂ fragment.